Biotechnology working with microoranisms

BiotechnologyWorking with Micro-organisms

Intermediate 25493

June 1999

BiotechnologyWorking with Micro-organisms

Intermediate 2

Support Materials

HIGHER STILL

INTERMEDIATE 2 BIOTECHNOLOGY

UNIT 2

WORKING WITH MICRO-ORGANISMS

STUDENT LABORATORY MANUAL

Biotechnology: Working with Micro-Organisms (Int 2) Student Laboratory Manual 1

UNIT 2: WORKING WITH MICRO-ORGANISMS (INT 2)

STUDENT LABORATORY MANUAL

ContentsGuide to CandidateMicrobiological procedures1: Preparation of work space2: Pouring plates3: Subculturing micro-organisms

3.1: Loop transfer of micro-organisms

3.1.1 solid to solid

3.1.2 liquid to solid

3.1.3 solid to liquid

3.1.4 liquid to liquid

3.2: Streak plate inoculations

3.2.1. solid to solid


3.3: Plate to plate subculture of fungal mycelium

3.4: Use of sterile swabs to sample the environment4: Separating micro-organisms5: Microscopic examination of micro-organisms

5.1: Magnification

5.2: Setting up a microscope

5.3: Microscopic examination of pond water/ hay infusion/yeast

5.3.1 Cavity slide preparation

5.3.2 Bright field microscopy

5.3.3 Dark field microscopy

5.3.4 Phase contrast microscopy

5.4: Calculation of specimen size using a microscope6: Staining

6.1: Preparation of a smear of bacteria or yeast from a solid culture

6.2: Observation of bacteria using the simple stain methylene blue

6.3: Observation of filamentous fungi using lactophenol blue

6.4: Negative staining of yoghurt with nigrosin

6.5: Staining of root nodules to observe Rhizobium

6.6: Vital staining of yeast with neutral red



GUIDE TO CANDIDATEThis unit of Intermediate 2 Biotechnology, ‘Working with Micro-organisms’ is apractical unit in which you will learn some techniques of handling and studyingmicro-organisms in the laboratory. These include plate pouring, subculturing micro-organisms, separating a mixed culture, staining and microscopy.

To achieve this unit, you must:• know and understand how microbiological, microscopy and staining techniques

are carried out in a given way; and• become competent in carrying out a range of microbiological and microscopy

techniques.

To achieve Outcome 1, you must pass a written end of unit assessment which will testyour knowledge and understanding of the theory behind the practical techniques. Theinformation you need for this is given in the introduction to each activity.

To achieve Outcome 2, you must become proficient in practical microbiologicaltechniques. You will have to practise them a number of times until you are familiarwith the procedures. Your teacher/lecturer will show you the techniques described inthis laboratory manual.

Each time you perform a technique, you must• prepare for the work as you have been shown by your teacher or lecturer• carry out the techniques safely and according to instructions• keep a record of the work you do• record your results and observations clearly.

You should keep the record of your work and your observations/results in a laboratorydiary/notebook and make an entry each time you carry out a piece of work or observeresults. The diary can be kept in a simple notebook or jotter and should be submittedas evidence for Outcome 2.

Each entry should have:• the date when the work was carried out• a heading• a brief description of the technique you carried out including incubation times and

temperatures if appropriate• observations or results recorded in an appropriate manner (words, diagram, table

etc)• discussion of results if appropriate.

An example of a diary entry is given below.


Example of laboratory diary entry.

1.6.98 Subculturing, liquid to solid.Micrococcus luteus labelled ‘AA, M luteus, 27.5.98’, was asepticallytransferred from a broth culture to an agar plate and plated out to obtainsingle colonies using the streak plate method. The plate was incubatedupside down at 30°C for 72 hours and then placed in a refrigerator till itcould be examined.

The technique was also carried out with Saccharomyces cerevisiae but itwas incubated for five days at 30°C.

8.6.98 Results

Organism Single colonies Contamination

M luteus Yes No

S cerevisiae Yes Yes

Discussion A green fungus contaminated the S cerevisiae plate. Aspore from the air possibly entered the plate during theprocess of plating out and developed into the fungalcolony when the plate was incubated. The contaminatedplate cannot be used for further subculture as sporesfrom the fungal colony may have contaminated the Scerevisiae colonies.


MICROBIOLOGICAL PROCEDURES

Introduction

The aim of this unit is to acquire some of the basic technical skills used in the study ofmicrobes.

In the practicals carried out in this unit, you will be working with living micro-organisms, mainly bacteria and fungi. Although the organisms you will be using areconsidered safe, there is always a small risk of contamination and so all organismsmust always be treated as if they could cause disease and you must follow therecommended safety procedures.

The aims of these safety procedures are:• to avoid bringing contaminating organisms to the laboratory bench• to avoid contaminating laboratory cultures• to avoid contaminating yourself, your colleagues and your surroundings• to avoid taking contaminating organisms out of the laboratory.

Good Working Practice

To achieve the above aims, you must develop good working practice by following theprocedures below and all further safety instructions throughout the unit.

General safety procedures in the laboratory

• Do not eat, drink or smoke in the laboratory.

• Do not lick fingers or labels, use only self adhesive labels.

• Avoid touching the face.

• Tie back long hair.

• Cover any cuts and grazes with a waterproof plaster.

• Wear a lab coat and keep it fastened.

• Speak quietly and avoid unnecessary movement around the laboratory.

• Report all accidents, no matter how trivial, to the teacher/lecturer.

• Do not pipette by mouth.

• Keep doors and windows closed.


On entering the microbiology laboratory

1. Hang up outdoor coats.

2. Leave bags at the side of the room taking only essentials to the workbench.

3. Wash hands thoroughly.

4. Dry hands using disposable paper towels.

5. Cover any wounds with a waterproof plaster.

6. Put on a lab coat.

7. Collect disinfectant and paper towel.

8. Swab bench with disinfectant.

You are now ready to start work. Do not touch your bag, outdoor wear etc.without first washing your hands. Do not leave the laboratory wearing a labcoat.

Bench Management

1. Swab bench with disinfectant at start and end of work using a paper towel.

2. Sit on the lab stool with legs under the bench.

3. Organise the equipment you are using so that it is within easy reach.

4. Keep bench as uncluttered as possible.

5. Do not lay contaminated materials on bench.

6. Report all spillages and breakage to the teacher or lecturer in charge.

Before leaving the microbiology laboratory


2. Place stool under bench.

3. Remove and put away lab coat.


5. Collect outdoor coat and leave laboratory.


1: PREPARATION OF WORK SPACE

Preparing yourself

When working with micro-organisms, it is important to develop what is known asaseptic technique so that you do not become contaminated with the micro-organismsyou are working with nor do you contaminate your cultures. Before starting work youmust wash your hands thoroughly with soap and water. This mechanically removesmany of the organisms on your skin. Drying with a fresh paper towel preventstransfer of organisms from a towel used by other people.

You should then put on a lab coat to protect your own clothes. In the event of a spilland contamination occurs, a lab coat can be carefully removed.

Preparing the work space

The surface on which you work must not absorb any fluid or have cracks which couldharbour micro-organisms. Some micro-organisms can survive for a considerablelength of time in the environment and if spilled on to a rough or absorbent surfacecould present a potential source of contamination both to others using the laboratoryand to cultures. If the bench surface is unsuitable, it should be covered withBenchkote or other non absorbent material.

Whatever the surface, before starting work you must swab your work space withdisinfectant to reduce the risk of contamination of your cultures. You must keep theworking area tidy and uncluttered in order to minimise the risk of accident. Haveonly the essentials on the bench.

While carrying out the experiments in this unit, you will use equipment with micro-organisms which will have to be disposed of safely. You should always have adisposal container of disinfectant within easy reach. If you have to stretch, there isa risk of knocking over apparatus or dropping organisms from a pipette for example.

A Bunsen burner is essential to the technique of aseptic transfer. You should place iton the bench so that you can reach it easily (the layout of equipment will be discussedlater). The Bunsen flame provides an updraught which carries air upwards away fromthe operator reducing the risk of contamination.

Air movement should be kept to a minimum in microbiology laboratories to preventcontamination. While practical work is being performed, doors and windows shouldbe kept shut.

In professional microbiology laboratories, specialised cabinets are used for thetransfer of micro-organisms for safety reasons. Transfer chambers are suchcabinets. The operator works with their hands and forearms in the chamber,transferring microorganisms, preparing sterile media etc. A lit Bunsen within thecabinet maintains an airflow into the cabinet so helps prevent contamination.


Lamina flow chambers or cabinets are more sophisticated types of transferchamber. They incorporate fans which control airflow and sometimes filters whichprevent any micro-organisms leaving the chamber. In cases where scientists areworking with extremely dangerous organisms, they work in specialised laboratorieswhose atmosphere is maintained at a pressure lower than that of its surroundings,preventing escape of laboratory air to the outside.

Fig. 1: Example of a transfer chamber


Questions: Preparation of workspace

1. State two things you must do before starting to prepare your work space.

2. Why must the surface of a work area be non-absorbent?

3. Give one reason for swabbing the work surface with disinfectant before and afteruse.

4. When setting out your work space, what precaution should you take to avoid therisk of knocking over equipment?

5. Explain why doors and windows in a microbiology lab should be kept shut.

6. Why should a microbiologist always carry out transfer work in the region of alighted Bunsen burner?


Activity 1: Preparation of workspace

Materials requiredMaterials required by each student:Benchkote and non-absorbent tape if required.Disinfectant and paper towelDiscard jar with disinfectantBunsen burnerWire loopCultures and media

InstructionsThe following applies to right-handed operators. Left-handed people should reversethe arrangement on the bench.

1. Tie back long hair.

2. Wash and dry hands thoroughly.

3. Cover any cuts/grazes with waterproof plaster.

4. Put on lab coat.

5. Collect the materials indicated above.

6. Attach benchkote to the bench if necessary using sticky tape.

7. Swab the surface of your work space with disinfectant using the paper towel.

8. Place your notes, pen etc. away from the immediate area in which you arecarrying out the work.

9. Place the wire loop to the right of the bench so that you can reach it with ease.

10. Place the Bunsen burner on a heat resistant mat centrally so that you can reach itwith ease but not so close that you are likely to burn yourself.

11. Place cultures and media to the left but still within easy reach. Place discard jarwith disinfectant to the right within easy reach.

12. In your laboratory diary/notebook, draw a labelled diagram of the bench set up.

13. Once you have completed an operation place the completed materials to yourright.

You must set up your bench in this way each time you carry out a microbiologypractical.


2: POURING PLATES

Micro-organisms are grown in or on a culture medium containing all the requirednutrients for growth. Culture media can be liquid or solid. Liquid or broth mediaalthough convenient have some disadvantages. Growths usually do not exhibitcharacteristic appearances in them and, except when they are designed for a specificbiochemical test, they are of limited use in identifying species. Also, contaminatingorganisms cannot be seen readily in liquid media and liquid media are more difficultto handle in ways which avoid formation of aerosols.

On solid media, micro-organisms grow to form discrete colonies (See Fig.2). Eachcolony is a clone of cells originating from a single organism and represents the growthof a single species. Micro-organisms can be identified by the appearance exhibitedby the different colonies.

Fig. 2: Bacterial colonies on a nutrient agar plate


AgarLiquid media can be made solid by the addition of agar. Agar has no nutritionalvalue nor does it inhibit growth, it is simply a solidifying agent.

The melting and solidifying points of agar are not the same . At the concentrationsnormally used, most agars melt at about 95°C when heated but solidify only whencooled to about 42°C. This low solidifying point allows heat-sensitive nutrients to bemixed with molten agar at temperatures as low as 45°C before pouring. The highmelting point ensures that the medium remains solid at all laboratory incubationtemperatures.

Pouring Agar PlatesAgar plates are prepared by pouring liquid agar at 55°C into sterile Petri dishes andallowing it to solidify.

Before pouring, unopened plates are labelled on the underside using an indeliblepen or wax pencil with initials, date and type of agar. This prevents confusion shouldlids inadvertently be swapped. The plates are then placed the right way up ready toreceive molten agar.

Sterile nutrient agar in universal bottles or other containers is first melted by heatingto 100°C then cooled in a water bath to 55°C. Using aseptic technique, a bottle neckis flamed and the agar poured gently into a Petri dish on a flat surface, raising the lidof the dish only far enough for the mouth of the bottle to enter. The lid is thenreplaced and the plate left undisturbed until the agar has cooled and set.

It is essential that the surface of the medium should be dry in order to maintainsingle colonies. Condensation produced from the cooling of the agar can make theagar surface wet but is normally reduced by pouring the agar at 55°C and if necessarythe plates can be dried open and upside down (see Fig.3) in an undisturbed area.

Any plates showing lumps, bubbles or growth should be discarded. Acceptableplates should be stored upside down until required (see Fig.4).

Plates are always incubated and stored in the inverted position to preventcondensation dropping on to the agar surface.

Fig.3: Drying of plate Fig.4: Storing of plates

Biotechnology Working with Micro-organisms (Int2) Student Laboratory Manual 13

Questions: Pouring plates

1. On what part of the plate do you write the label?

2. Name the three details you should record in the label.

3. What precaution do you take before pouring the agar?

4. How do you avoid bubbles on the surface of the agar?

5. How do you prevent excess condensation gathering on the lid of the plate after theagar is poured?

6. Where and how should the plates be stored?

7. Name the four characteristics well poured plates should possess.


Activity 2: Pouring plates

Materials required

Materials required by each student:Lab coatEye protectionDisinfectant and paper towelsDiscard jar with disinfectant3 bottles of sterile nutrient agar3 sterile plastic Petri dishesBunsen burner and mat

Materials to be shared:Benchkote if necessaryWater bath with water at 55°CFine tipped indelible marker pensAutoclavable bags for disposal of contaminated plates

Instructions

These instructions are for right-handed people. If you are left handed, please reversethe instructions accordingly.

1. Wear a lab coat and use eye protection.

2. Collect the Petri dishes, Bunsen burner and mat.

3. Label the empty sterile Petri dishes on the base with name, date and type of agar.

(N.B. if the lid comes off, the plate is no longer sterile and you must discard it).

4. Light the Bunsen burner.

5. Collect one bottle of sterile molten agar from the water bath. Check it is not too

hot and that it has not started to solidify.

6. Place a Petri dish right way up on the bench.

7. Check that the top of the bottle of agar is loose.


8. Hold the bottle of Agar in the left hand.

9. Unscrew and remove the cap of the bottle with the little finger of the right hand.

10. Pass the neck of the bottle backwards and forwards through a blue Bunsen flame.

11. With the right hand, lift the lid of the Petri dish a little and gently pour in the

molten agar.

For copyright reasons, the image that appeared on this page in print is

unavailable.


12. Replace the lid of the Petri dish.

13. Replace the cap of the bottle and put it down.

14. Swirl the plate very gently to distribute the agar evenly. (N.B. The agar must not

touch the lid of the plate and must have a smooth surface with no bubbles).

15. Repeat for the other 2 bottles and plates.

16. Leave the agar to solidify.

17. Once cool, turn the plates upside down.

18. Record briefly the procedure you have carried out in your lab diary/notebook.

19. After a few days, examine your plates. Record your observations in a table. For

each plate, make a note of the following characteristics in your lab

diary/notebook:

• smooth surface to agar?

• even layer of agar?

• base of Petri dish covered?

• contamination?


Unit 2: Working with Micro-organisms (Int 2)

Title: Pouring platesName of candidate_____________________________________________________

PERFORMANCE CRITERIA GUIDANCE ACHIEVED

a. Preparation for work is inaccordance with givenspecifications.

• Hands washed

• Protective clothing worn

• Workspace prepared appropriately.

b. Techniques are carried outin accordance with safepractice and givenspecifications.

• Plates labeled on underside

• Agar cooled to pouring temperature

• Aseptic technique satisfactory

• Plates dried

• Plates incubated.

c. Record of work is clearand accurate.

Entry in lab diary/notebook:

• Date work carried out

• Heading

• Brief description of technique includingincubation times and temperatures.

d. Results and relevantobservations are reportedclearly.

Comments on characteristics of plates:

• Smooth surface to agar

• Even layer of agar

• Base of Petri dish covered

• Contamination.

Teacher/lecturer’s signature:

Date:



3: SUBCULTURING MICRO-ORGANISMS

In the laboratory, micro-organisms are usually grown or cultured in liquid medium(broth) or on solid medium (agar plates or slopes). Growth of bacteria and yeastsshows as cloudiness or turbidity in the broth although sometimes bacteria grow as alayer on the surface of the broth or at the bottom of the culture tube.

The growth on plates depends upon how the plate has been inoculated. (Fig. 5).

Subculturing is the aseptic transfer of micro-organisms from a culture to freshmedium. The freshly inoculated medium is then incubated at the temperatureappropriate for growing the organism.

There are four subculturing procedures with which you should become familiar. Theyare:• solid to solid: the transfer of bacteria or fungi from an agar slope or plate culture

to an agar plate• solid to liquid: the transfer of bacteria or fungi from an agar slope or plate culture

to a broth• liquid to solid: the transfer of bacteria or fungi from a broth culture to an agar

slope or plate• liquid to liquid: the transfer of bacteria or fungi from a broth culture to a broth.

Containers of culture media to be inoculated must be labelled with initials, date andname of organism. To prevent possible confusion, plates are marked on the undersidewhile tubes and bottles must be labelled on the side. Lids are not labelled.

The use of aseptic technique minimises the risk of contamination of cultures and alsoreduces the risk of micro-organisms from the laboratory cultures escaping to theenvironment.

simple streak plate(smear plate)

streak plate

Fig. 5: Types of inoculations on agar plates


You will use a wire loop, a straight wire, forceps or a scalpel to subculture bacteria orfungi or to transfer specimens to a slide. The wire loop is the basic tool of themicrobiologist and you should learn to handle it correctly.

Flame sterilisation of instruments

Metal instruments used to transfer micro-organisms are sterilised using red heat in aBunsen burner before and after use. They must be heated till red hot to make surethat any contaminating bacterial spores are destroyed.

Good flaming technique is very important to avoid contamination of thesurrounding air with aerosols.

A Bunsen burner is lit and the air hole opened fully to provide a blue flame. Theoperator holds the loop between thumb and fingers as if holding a pencil very loosely,at an angle that is almost vertical. (Fig. 6)

Fig. 6: Holding a wire loopFor copyright reasons, this image is unavailable


The loop is placed in the light blue cone of the flame (Fig. 7). Positioning the loop inthis cool area of the flame allows any liquid to dry out and prevents formation ofaerosols. Aerosols are fine liquid or solid particles that are dispersed into the air andmight contain micro-organisms.

Fig. 7: Placing a loop in blue cone of flameFor copyright reasons, this image is unavailable

After any liquid material has evaporated, the loop is drawn slowly up into the hottestregion of the flame (immediately above the light blue cone) and held there until it isred hot. (Fig. 8)

Fig. 8: Drawing loop into hottest region of flameFor copyright reasons, this image is unavailable


The loop is then withdrawn from the flame and allowed to cool before touchingmicroorganisms. (Fig. 9)

Fig. 9: Cooling the loopFor copyright reasons, this image is unavailable

Straight wires may be sterilised in the same way.

Aseptic transfer operations

Once the loop has been sterilised and cooled, it is used to remove organisms (theinoculum) from a culture and inoculate sterile growth medium. When working withmicro-organisms, aseptic techniques are used to avoid escape of organisms to thesurroundings and contamination of the culture by micro-organisms from theenvironment.

To minimise the chances of contamination, cultures and media are exposed to the airfor the minimum time it takes to perform a manipulation or to make observations. Allsubculturing procedures are carried out close to a Bunsen flame.

After flaming, the loop is not put down until the procedure has been completed.

Lids of Petri dishes are never completely removed. They are opened just enough toallow entry of the loop to perform the manipulation and minimise exposure to the air.

Liquid cultures are disturbed as little as possible to reduce the risk of aerosolformation. Lids from cultures are never placed on the bench surface wherecontamination might occur. They are removed from the bottle or tube using the littlefinger, held there while manipulation of the culture takes place and then replaced.The rest of the hand is free to carry out the manipulation. Lids are tightened to preventspillage before incubation.


To remove any contaminating organisms on the neck of a bottle, it is passed througha hot Bunsen flame on removal and before replacement of the lid.

The loop must be flamed to red heat when the subculturing procedure isfinished.

The table below summarises potential points of contamination and the techniquesemployed to minimise the risk.

Contamination risk Precaution

Inoculating loop Flame and cool.Do not lay down loop until procedure iscomplete.Work close to Bunsen flame.

Opening of Petri dish (solid medium) Open lid for as short a time as possible.Open lid just enough to insert wire loop.

Opening of liquid cultures (bottles, tubes) Hold the lid in crook of little finger –never place on work bench.Pass neck through a hot Bunsen flamebefore insertion and after withdrawal ofloop to kill any contaminating organisms.

Incubation of cultures

After inoculation, cultures are incubated at a given temperature. Petri dishes shouldbe placed upside down to prevent condensation dropping on to cultures. The lids ofPetri dishes should be secured to the base with diagonal strips of sellotape. Bottlesshould be stored upright in a container which will prevent them being knocked over.

Subculture results

Subculturing has been successful when the transferred organisms have grown in thenew medium without contamination.

Contaminating organisms can usually be observed on plate cultures as single colonieswith a different appearance. In a broth culture, it is impossible to tell by simplyobserving the broth whether the culture is pure. The liquid culture must be streakedout on an agar plate to determine if there are any contaminants.

In the practical work associated with this course, you will generally be working withknown organisms and with pure cultures. A pure culture is one in which there isonly one type of organism.



When working with micro-organisms, you must ‘think safety’ all the time andconstantly be aware of points at which contamination might occur and takemeasures to minimise the risks.

Subculturing procedures

In order to achieve aseptic transfer of micro-organisms, there are a number ofprocedures that must be followed. These techniques will be demonstrated.

• Inoculating loops must be sterilised by flaming before and after use see ‘Flame

sterilisation of instruments).

• Once flamed, the loop must never be put down but held in the hand and allowed to

cool. Inoculating instruments should not be waved around.

• The necks of tubes and bottles must be flamed after removing and before

replacing a lid.

• Caps of bottles must never be put down.

• Cultures should be exposed to the air for as short a time as possible.

• Lids of Petri dishes should be raised only enough to admit the wire loop and

permit the manipulation.

Disposal of Contaminated Materials

It is essential that contaminated materials be disposed of safely and without risk toanyone. Cultures and contaminated equipment such must be sterilised beforedisposal. Technical staff will carry out these procedures.

You will be directed as to where to place materials for disposal. Make sure that youfollow instructions for disposal of contaminated equipment and materials.

In the event of a spill, you must always inform the teacher/lecturer or

technician.


Questions: Subculturing micro-organisms

1. Describe the correct way to hold a wire loop.

2. When flaming a wire loop, the loop should first be placed in the pale blue cone ofthe Bunsen flame. Why?

3. How do you judge when the loop is sterilised?

4. Why must the loop be cooled before subculturing?

5. State two precautions that should be taken to prevent contamination of liquidmedia and cultures.


6. What process must be carried out at the end of every subculturing procedure usinga wire loop?

7. Explain why the lids of Petri dishes and bottles should not be used to labelcultures.

8. What three pieces of information should always be used on culture labels?

9. Why is it important to incubate or refrigerate plate cultures upside down?

10. Why should cultures be sterilised before final disposal?


Activity 3.1.1: Loop transfer, solid to solid

Materials requiredMaterials required by each student:Lab coatBunsen burner and matDisinfectant and paper towelsDiscard jar with disinfectant1 plate culture Micrococcus luteus1 plate culture Saccharomyces cerevisiae (yeast)Wire loop4 plates of sterile nutrient agar

Materials to be shared:Benchkote if necessaryIncubator at 30°CAutoclavable bags for disposal of contaminated plates



InstructionsThese instructions are for right-handed people. If you are left handed, please reversethe instructions accordingly.

1. Wear a lab coat.

2. Prepare your work space on the bench, collect the materials and set them out

correctly on the bench.

3. Label the bases of the Petri dishes containing sterile nutrient agar with initials,

date, name of microorganism and s/s (solid to solid).

4. Turn plate upright.

5. Hold the loop in the right hand.

6. Flame the loop and allow to cool. Do not put down loop or wave it around.

7. Lift the lid a little of the Petri dish containing the inoculum with the left hand.

8. Touch a single colony with the wire loop.

9. Withdraw loop. Do not put down loop or wave it around.

10. Replace lid of Petri dish.

11. Partially lift the lid of the Petri dish containing the solid medium.

For copyright reasons, the image that appeared on this page in print is

unavailable


13. Replace the lid of the Petri dish.

14. Flame loop, and place on heat resistant mat.

15. Secure the lid of the Petri dish to the base with sellotape.

16. Incubate plate upside down at 30°C for 72 hours.

17. Repeat for the other inocula.

Record briefly the procedure you have carried out in your lab diary/notebook. Afterincubation, take a note of your observations. These could include a diagram and/ortable and should include the following:

• whether growth is present• a description of the growth• whether the plate is contaminated.

12. Holding the loop parallel with the surfaceof the agar, smear the inoculum across themedium as shown.




Title: Subculturing micro-organismsName of candidate_____________________________________________________



• Hands washed



b. Techniques are carried out inaccordance with safe practiceand given specifications.

• Instructions followed accurately


• Technique carried out in accordancewith safe practice

• Inoculating instrument sterilisedcorrectly

• Removal of inoculum satisfactory

• Inoculation method satisfactory

• Plates/ broths incubated appropriately

• Satisfactory growth

• No growth if inoculated with sterilewater.

c. Record of work is clear andaccurate.



• Heading



Comments on:

• Whether growth is present

• Description of growth

• Contamination.

Organism:Type of inoculation:Teacher/lecturer’s signature:

Date:


Activity 3.1.2: Loop transfer, liquid to solid

Materials requiredMaterials required by each student:Lab coatEye protectionBunsen burner and matDisinfectant and paper towelsDiscard jar with disinfectant1 broth culture Micrococcus luteus1 broth culture Saccharomyces cerevisiae (yeast)Wire loop4 plates of sterile nutrient agar






2. Prepare your work space on the bench, collect the materials and set them outcorrectly on the bench.

3. Label the underside of plates with initials, date, name of microorganism and l/s(liquid to solid).

4. Turn the plate upright.5. Loosen the tops of the universals containing the broth cultures so that they can be

removed easily.6. Hold the loop in the right hand.7. Flame the loop and allow to cool. Do not put down loop or wave it around.8. Lift the universal containing the inoculum with the left hand.9. Remove the lid of the universal with the little finger of the right hand. Do not put

down the lid.

For copyright reasons, the images that appeared on this page in print areunavailable.


10. Flame neck of universal.11. Insert the loop into the culture broth (bring bottle to loop, not loop to the bottle)

and withdraw. Take care not to touch the sides of the bottle or its mouth.12. Flame the neck of the universal.



13. Replace the lid on the universal using the little finger. Turn the bottle, not the lid.14. Place the universal on the bench.15. With the left hand, partially lift the lid of the Petri dish containing the solid

medium.

17. Replace lid of Petri dish.18. Flame loop and place on heat resistant mat.19. Secure the lid to the base with diagonal strips of sellotape.20. Incubate plates upside down at 30°C for 72 hours.21. Repeat for the other inocula.


• whether growth is present• a description of the growth• whether there are isolated single colonies• whether the plate is contaminated.

16. Holding the loop parallel with the surfaceof the agar, smear the inoculum across themedium as shown.






e. Preparation for work is inaccordance with givenspecifications.

• Hands washed



f. Techniques are carried out inaccordance with safe practiceand given specifications.










g. Record of work is clear andaccurate.



• Heading


h. Results and relevantobservations are reportedclearly.

Comments on:



• Contamination.


Date:


Activity 3.1.3: Loop transfer, solid to liquid

Materials requiredMaterials required by each student:Lab coatEye protectionBunsen burner and matDisinfectant and paper towelsDiscard jar with disinfectant1 plate culture Micrococcus luteus1 plate culture Saccharomyces cerevisiae (yeast)Wire loop4 universals of sterile nutrient broth








3. Label the universals containing sterile nutrient broth with initials, date, name of

microorganism and s/l (solid to liquid).

4. Loosen the tops of all the universals so that they can be removed easily.

5. Hold the loop in the right hand.

6. Flame the loop and allow to cool. Do not put down loop or wave it around.

7. Lift the lid slightly of the Petri dish containing the inoculum with the left hand.

8. Touch a single colony with the wire loop.

9. Withdraw loop. Do not put down loop or wave it around!


11. Lift a universal of sterile nutrient broth in the left hand.

12. Remove the lid of the universal with the little finger of the right hand which still

holds the charged wire loop. Do not put down the lid.

For copyright reasons, the imagse that appeared on this page in print are

unavailable


13. Flame the neck of the uinversal.14. Insert the loop charged with inoculuminto the sterile broth (bring the bottle to the

loop), agitate gently, touch on the inside of the universal and withdraw.



15. Flame the neck of the universal.16. Replace the lid on the universal using the little finger of the right hand. (Turn the

bottle, not the lid.)17. Place universal on bench.18. Flame the loop and place on a heat resistant mat.



19. Tighten lid of universal to make secure but do not overtighten.

20. Incubate inoculated universal at 30°C for 72 hours.

21. Repeat once with the same inoculum and twice with a second culture as the

inoculum.


• whether growth is present• a description of the growth (heavy or light, spread evenly throughout broth or

concentrated at top or foot etc.).





i. Preparation for work is inaccordance with givenspecifications.

• Hands washed



j. Techniques are carried out inaccordance with safe practiceand given specifications.










k. Record of work is clear andaccurate.



• Heading


l. Results and relevantobservations are reportedclearly.

Comments on:



• Contamination.


Date:


Activity 3.1.4: Loop transfer, liquid to liquid

Materials requiredMaterials required by each student:Lab coatEye protectionBunsen burner and matDisinfectant and paper towelsDiscard jar with disinfectant1 broth culture Micrococcus luteus1 broth culture Saccharomyces cerevisiae (yeast)Wire loop4 universals of sterile nutrient broth2 bottles sterile water

Materials to be shared:

Benchkote if necessaryIncubator at 30°CAutoclavable bags for disposal of contaminated plates





1. Wear a lab coat and use eye protection2. Prepare your work space on the bench, collect the materials and set them out

correctly on the bench.3. Label the universals containing sterile nutrient broth with initials, date, name of

microorganism and l/l (liquid to liquid).4. Loosen the tops of all the universals so that they can be removed easily.5. Hold the loop in the right hand.6. Flame the loop and allow to cool. Do not put down loop or wave it aroound.

For copyright reasons, the image that appeared on this page in print isunavailable.


7. Lift the universal containing the inoculum with one hand.8. Remove the lid of the universal with the right hand. Do not put down the lid.9. Flame neck of universal.10. Insert the loop into the culture broth and withdraw (bring bottle to loop; not loop

to bottle). Take care not to touch the sides of the bottle or its mouth.



11. Flame the neck of the universal.12. Replace the lid on the universal using the little finger. Turn the bottle, not the lid.13. Place the universal on the bench.14. Lift a universal of sterile nutrient broth in the left hand.15. Remove the lid of the universal with the little finger of the right hand which still

holds the charged wire loop. Do not put down the lid. Turn the bottle, not the lid.16. Flame the neck of the universal.



17. Insert the loop charged with inoculum into the sterile broth (bring bottle to loop.)Agitate gently, touch on the inside of the universal and withdraw.

18. Flame the neck of the universal.



19. Replace the lid on the universal using the little finger of the right hand. Turn thebottle, not the lid.

20. Place universal on bench.21. Flame the loop and place on a heat resistant mat.22. Tighten lid of universal to make secure but do not overtighten.23. Repeat once with the other inoculum and twice more using sterile water as the

inoculum to check aseptic technique.24. Incubate the inoculated universals at 30 °C for 72 hours.

For copyright reasons, the images that appeared on this page in print are

unavailable.

Record briefly the procedure you have carried out in your lab diary/notebook. . Afterincubation, take a note of your observations. These could include a diagram and/ortable and should include the following:

• whether growth is present• a description of the growth (heavy or light, spread evenly throughout broth or

concentrated at top or foot etc.).






m. Preparation for work is inaccordance with givenspecifications.

• Hands washed



n. Techniques are carried out inaccordance with safe practiceand given specifications.










o. Record of work is clear andaccurate.



• Heading


p. Results and relevantobservations are reportedclearly.

Comments on:



• Contamination.


Date:



The term ‘streaking out’ or ‘plating out’ is applied to the inoculation of solid mediain Petri dishes using a technique involving successive strokes with a wire loop. Theaim of the procedure is to obtain isolated single colonies. The loop is charged with asmall amount of culture and several strokes in series are then made on the surface ofthe medium with the aim of diluting the bacteria so that single cells are distributed onthe surface. The loop is flamed between strokes to reduce the number of organismsand increase the chance of isolating single cells. On incubation, each single celldivides successively to produce a single colony. In this way, pure cultures can beobtained. (Fig.10)

Fig.10: Streak plate showing single colonies


Questions: Streak plate inoculations

1. Why do microbiologists use the technique of ‘streaking out’?

2. When performing ‘streaking out’, the loop is flamed between strokes. Why?

3. What is meant by the term ‘pure culture’?

4. How is a bacterial colony formed?


Activity 3.2.1: Streak plate inoculation, solid to solid

Materials requiredMaterials required by each student:Lab coatBunsen burner and matDisinfectant and paper towelsDiscard jar with disinfectant1 plate culture Micrococcus luteus1 plate culture Saccharomyces cerevisiae (yeast)Wire loop4 plates of sterile nutrient agar

Materials to be shared:Benchkote if necessaryIncubator at 30°CAutoclavable bags for disposal of contaminated platesFine tipped marker pens



Instructions

These instructions are for right-handed people. If you are left handed, please reversethe instructions accordingly.

1. Wear a lab coat.2. Prepare your work space on the bench, collect the materials and set them out

correctly on the bench.3. Label the bases of the Petri dishes containing sterile nutrient agar with initials,

date, name of microorganism and s/s.4. Hold the loop in the right hand.5. Flame the loop and allow to cool. Do not put down loop or wave it around.6. Lift the lid a little of the Petri dish containing the inoculum with the left hand.7. Touch a single colony with the wire loop.8. Withdraw loop. Do not put down loop or wave it around!9. Replace lid of Petri dish.10. Partially lift the lid of the Petri dish containing the solid medium.

For copyright reasons, the imagethat appeared on this page in print is

unavailable.


12. Flame loop and allow to cool.

14. Flame the loop and allow to cool.


11. Holding the loop parallel with the surfaceof the agar, smear the inoculum backwardsand forwards across a small area of themedium

13. Turn plate. Streak loop from A across thesurface of the agar in three parallel lines.

A

15. Turn plate. Streak loop from B across thesurface of the agar in three parallel lines.

A

B


18. Replace lid of Petri dish.19. Flame loop and place on heat resistant mat.20. Repeat for second inoculum.21. Incubate plate upside down at 30°C for 72 hours.

Record briefly the procedure you have carried out in your lab diary/notebook. After

incubation, take a note of your observations. These could include a diagram and/or

table and should include the following:


17. Turn plate. Streak loop from C to D acrossthe surface of the agar as shown.

A

B

C

D






q. Preparation for work is inaccordance with givenspecifications.

• Hands washed



r. Techniques are carried out inaccordance with safe practiceand given specifications.










s. Record of work is clear andaccurate.



• Heading


t. Results and relevantobservations are reportedclearly.

Comments on:



• Contamination.


Date:


Activity 3.2.2: Streak plate inoculation, liquid to solid

Materials requiredMaterials required by each student:Lab coatEye protectionDisinfectant and paper towelsDiscard jar with disinfectantBunsen burner and mat1 broth culture Micrococcus luteus1 broth culture Saccharomyces cerevisiae (yeast)Wire loop4 plates of sterile nutrient agar





1. Wear lab coat and eye protection.2. Prepare your work space on the bench, collect the materials and set them out

correctly on the bench.3. Label the underside of plates with initials, date name of microorganism and l/s.4. Turn the plate upright.5. Loosen the tops of the universals containing the broth cultures so that they can be

removed easily.6. Hold the loop in the right hand.7. Flame the loop and allow to cool. Do not put down loop or wave it around.8. Lift the universal containing the inoculum with the left hand.9. Remove the lid of the universal with the right hand. Do not put down the lid.10. Flame neck of universal.



11. Insert the loop into the culture (bring bottle to\loop, not loop to bottle) andwithdraw. Take care not to touch the side of the bottle or its mouth.

12. Flame the neck of the universal.13. Replace the lid on the universal using the little finger. Turn bottle, not lid.14. Place the universal on the bench.15. With the left hand, partially lift the lid of the Petri dish containing the solid

medium.




19. Flame the loop and allow to cool.


16. Holding the loop parallel with the surfaceof the agar, smear the inoculum backwardsand forwards across a small area of themedium

18. Turn plate. Streak loop from A across thesurface of the agar in three parallel lines.

A

20. Turn plate. Streak loop from B across thesurface of the agar in three parallel lines.

A

B

22. Turn plate. Streak loop from C to D acrossthe surface of the agar as shown.

A

B

C

D


23. Replace lid of Petri dish.24. Flame loop and place on heat resistant mat.25. Repeat for second inoculum.26. Incubate plate upside down at 30°C for 72 hours.







u. Preparation for work is inaccordance with givenspecifications.

• Hands washed



v. Techniques are carried out inaccordance with safe practiceand given specifications.










w. Record of work is clear andaccurate.



• Heading


x. Results and relevantobservations are reportedclearly.

Comments on:



• Contamination.


Date:



It is sometimes difficult to pick up fungal mycelium with a wire loop or straight wire.Plate to plate inoculation is therefore often carried out using a sterile scalpel.

The scalpel is first sterilised by covering the blade in ethanol, lighting it in a Bunsenflame and allowing it to burn off. It is then used to cut through the fungal myceliumand agar below to extract a small block. This is then transferred aseptically to sterileagar and incubated. The wide end of a sterile Pasteur pipette or a cork borer flamed inethanol may also be used for the transfer operation.

Questions: Plate to plate subculture of fungal mycelium

1. What is meant by ‘fungal mycelium’?

2. Why is a scalpel used to subculture fungal mycelium?

3. Describe one difference apart from the use of a scalpel between subculturingfungal mycelium and subculturing bacteria from solid to solid?


Activity 3.3: Plate to plate subculture of fungal mycelium

Materials requiredMaterials required by each student:Lab coatEye protectionBunsen burner and matDisinfectant and paper towelsDiscard jar with disinfectant1 plate culture Penicillium roquefortii or Mucor heimalisSmall volume of ethanol in a covered beakerScalpel2 plates of sterile malt agar





1. Wear lab coat and use eye protection.



3. Label the bases of the Petri dishes containing sterile malt agar with initials, date,

name of micro-organism.

4. Hold the scalpel in the right hand.

5. Keep the ethanol at a safe distance from the Bunsen flame.

6. Dip the scalpel blade in ethanol, place in Bunsen flame briefly and allow ethanol

to burn off. Do not put down scalpel.

7. Lift the lid a little of the Petri dish containing the inoculum with the left hand.

8. Using the tip of the scalpel blade cut a square shape in the fungal mycelium (Fig.

11). This may be at the edge of the colony if there is heavy spore production.

9. Insert the blade under the square you have cut and lift a cube of agar with fungal

mycelium on top on to the blade of the scalpel.

10. Withdraw scalpel from Petri dish. Do not put down scalpel or wave it around!


12. Partially lift the lid of the Petri dish containing the sterile malt agar medium.

13. Place the cube of agar on your scalpel blade on to the centre of the sterile agar

(Fig.11). It does not matter whether it is upright or upside down.

14. Withdraw scalpel.

15. Dip the scalpel blade in ethanol, flame as above and place on heat resistant mat.

16. Incubate plate upside down at 30°C for 72 hours or at room temperature for 5

days..

17. Repeat for second inoculum.

Record briefly the procedure you have carried out in your lab diary/notebook. Afterincubation, take a note of your observations. These could include a diagram and/ortable and should include the following:• whether growth is present• a description of the growth• whether the plate is contaminated.


Fig.11: Transferring a block of agar with mycelium.

Source of inoculum Inoculum placed on sterile agar






y. Preparation for work is inaccordance with givenspecifications.

• Hands washed



z. Techniques are carried out inaccordance with safe practiceand given specifications.










aa. Record of work is clear andaccurate.



• Heading


bb. Results and relevantobservations are reportedclearly.

Comments on:



• Contamination.


Date:


3.4: Use of sterile swabs to sample the environment

The numbers and types of bacteria and fungi present on a surface such as thelaboratory bench can be estimated by swabbing the area with a sterile swab or cottonwool bud which has been dampened with sterile water. The swab is then rubbedacross a sterile agar plate which is then incubated.

Plates must be sealed with tape and not opened after incubation since the organismsthat grow are unknown.

Swabs are not sterilised in the Bunsen flame. You may receive them pre-sterilised,wrapped in aluminium foil or you may use commercial ones directly.

Questions: Use of sterile swabs to sample the environment

1. Why are swabs not sterilised in a Bunsen flame?

2. What precaution must you take when removing a swab from its sterile pack?

3. Why must you seal plates inoculated with swabs of the environment beforeincubation and dispose of them unopened?


Activity 3.4: Use of sterile swabs to sample the environment

Materials requiredMaterials required by each student:Lab coatBunsen burner and matSterile swabsDisinfectant and paper towelsDiscard jar with disinfectant1 plates of sterile malt agarI plate sterile nutrient agarBijoux bottle of sterile water

Materials to be shared:Benchkote if necessaryIncubator at 30°CAutoclavable bags for disposal of contaminated platesFine tipped marker pensSellotape




1. Wear a lab coat.



3. Label the bases of the Petri dishes containing sterile agar with initials, date and

area to be sampled.

4. Taking care not to touch the cotton wool end, remove a sterile swab from the pack

and hold it in your right hand.

5. Lift a bottle of sterile water in the left hand. Do not put down swab.

6. Remove the lid of the bottle with the little finger of the right hand which is still

holding the swab.

7. Flame the neck of the bottle.

8. Insert and withdraw the cotton wool bud.

9. Flame the neck of the bottle and replace the lid. Do not put down swab or wave it

around!

10. Rub the swab on the area to be tested as directed by your teacher/lecturer for 10 –

15 seconds.

11. Partially lift the lid of the agar plate and gently rub the swab across the surface of

the agar as shown. Take care not to break the agar

surface.


13. Sellotape lid to base of plate with diagonal strips.

14. Incubate plate upside down at room temperature for 72

hours.

15. Repeat for second inoculum.

Record briefly the procedure you have carried out in yourlab diary/notebook. After incubation, take a note of yourobservations. These could include a diagram and/or tableand should include the following:

• whether growth is present• a description of the growth.





cc. Preparation for work is inaccordance with givenspecifications.

• Hands washed



dd. Techniques are carried out inaccordance with safe practiceand given specifications.










ee. Record of work is clear andaccurate.



• Heading


ff. Results and relevantobservations are reportedclearly.

Comments on:



• Contamination.


Date:



4: SEPARATING MICRO-ORGANISMS

When professional microbiologists isolate micro-organisms from the environment oran infected person, it is extremely rare to obtain a pure culture. It is thereforenecessary to separate micro-organisms. Plating or streaking (see Fig. 10) can be usedto achieve this. Using isolated single colonies as inocula for further streak plates,pure cultures can be obtained.

Streaking out a mixed broth culture on an agar plate and incubating it to obtain singlecolonies of different types of bacteria or yeasts can simulate this.

Fig. 10: Streak plate showing individual colonies


Questions: Separating micro-organisms

1. What is meant by a ‘mixed culture’?

2. Explain why the procedure of streaking out can be used to separate micro-organisms in a mixed culture.

3. Describe how you could obtain pure cultures on agar plates from a mixed brothculture.


Activity 4: Separating micro-organisms

Materials requiredMaterials required by each student:Lab coatBunsen burner and matDisinfectant and paper towelsDiscard jar with disinfectant1 mixed broth culture of Phaffia (a red yeast) and Saccharomyces cerevisiae (acreamy white yeast)Wire loop2 plates of sterile malt agar

Materials to be shared:Benchkote if necessaryAutoclavable bags for disposal of contaminated platesFine tipped marker pens


1. Wear a lab coat.



3. Label the bases of the Petri dishes containing sterile malt agar with initials, date,

name of culture.

4. Using the mixed broth culture as the inoculum, streak out applying the method

you have used previously.

5. Incubate the plates at room temperature for 48 hours.

6. Repeat for second plate.

Record briefly the procedure you have carried out in your lab diary/notebook. Afterincubation, take a note of your observations. These could include a diagram and/ortable and should include the following:• whether growth is present• a description of the growths• whether there are isolated single colonies of each type of organism.

If you have obtained well isolated single colonies, use these as inocula for furtherstreak plates using the method you have used previously to obtain pure cultures.




Title: Separating a mixed cultureName of candidate_____________________________________________________



• Hands washed




• Loop/scalpel flamed correctly



• Plates incubated appropriately

• Single colonies of both types present

• Contaminated materials disposed ofappropriately

• Surface swabbed with disinfectant.




• Heading



Comments on:


• Description of growths

• Whether there are isolated single colonies ofeach type of organism.

Organisms:


Date:


5: Microscopic examination of micro-organisms

Micro-organisms are so small that they you cannot see them without the aid of amicroscope. Microscopes are used to produce an enlarged image of objects too smallto be seen with the naked eye.

Although microscopes produced by different manufacturers may look quite unlikeeach other, they all work on the same principle and consist essentially of similarworking parts.

To obtain the clearest image of micro-organisms using a microscope, you must learnhow to set it up properly. You must first know the principles on which themicroscope works.

Fig. 12: Structure of the microscopeFor copyright reasons, this image on is unavailable.


Part Description

Eyepiece lens It fits into the body tube. It contains lenses which magnify the

image produced by the objective lens and produces the image

the operator sees. Eyepiece lenses usually have a magnification

of x10. You may use x15 to observe bacteria.

Objectives Contain lenses which magnify the specimen. Objective lenses

bear a coloured ring which indicates their order of

magnification. Microscopes usually have objective lenses of

power x10 and x40. They may also have an oil immersion

objective (x90 or x100).

Stage A flat platform on which the specimen is placed. It has a hole

in the centre which allows light to pass up through the

specimen. There are usually two side clips which hold the slide

in position.

Condenser Contains lenses which focus the light into a cone. A condenser

focus control allows the condenser to be moved up and down so

that light is focused at the specimen. The condenser may not be

present in some microscopes.

Iris diaphragm The iris diaphragm can be opened or closed to control the angle

of the cone of light passing through the condenser. It makes the

specimen brighter and the image clearer.

Mirror Can be adjusted to reflect light up on to the specimen. One side

of the mirror is flat, the other is concave. The flat side is used

if a condenser is present. The mirror may be replaced with an

electrical light source.


Principles of the light microscope• Visible light passes through a substage condenser which focuses the light into a

sharp cone.• The light passes through the opening in the stage into the slide illuminating the

specimen.• The light passes through the objective lens and forms a magnified image of the

specimen which is usually darker than the background.• The eyepiece lens magnifies this image further and creates the image that the user

sees.

Oil immersionTo observe bacteria clearly with a microscope, it is usually necessary to use anobjective lens of x100. The lens must be immersed in a drop of oil which is placed onthe slide. This helps direct more light into the objective lens.

Questions: Structure and function of the microscope

1. What is the purpose of the microscope?

2. What part of the microscope controls the angle of light that passes up through thecondenser?

3. State the function of the stage on a microscope.

4. Name three parts of the microscope that contain lenses.

5. Why would you adjust the focus controls?


5.1: Magnification

The magnifying power is the degree of enlargement; that is, the number of times theimage appears greater than the original specimen.

To calculate this, multiply together the separate magnifying powers of the objectiveand eyepiece lenses. For example, the total magnification of a microscope fitted witha x10 eyepiece lens and a x10 objective lens is 10 x 10 = x100. Similarly, if a x40objective lens was being used on the same microscope, the total magnification wouldbe 10 x 40 = x400.

Questions: Magnification

Complete the following table.

Eyepiece lensmagnification

Objective lensmagnification

Totalmagnification

x15 x10 x150

x10 x10

x40 x400

x10 x1000

x15 x40


Activity 5.2: Setting up a microscope

Microscopes are expensive. Please treat them with care and make sure youclean them before putting them away.

MaterialsMaterials required by each studentMicroscopeBench lampPrepared slides of micro-organismsLens tissue

Instructions

1. Clean the lenses and all other glass surfaces on the microscope with lens tissue to

remove dirt and greasy marks as you have been shown.

2. Feel beneath the stage for the iris diaphragm control and set it to about half open.

3. Turn the condenser focus control and note the movement of the condenser. Raise

the condenser fully, then lower 1 – 2mm. Focus the condenser as shown by your

teacher/lecturer.

4. Place the slide on the stage, specimen uppermost and manoeuvre the slide until the

specimen is above the light source.

5. Turn the x 10 objective lens till it clicks into place above the slide.

6. Switch on the bench lamp and place it about 20cm from the microscope. Shine

the light directly on to the plane surface of the mirror, not the concave.

7. Turn the rough focus knob till the objective lens is as close as possible to the

stage.

8. Look down the eyepiece lens and slowly turn the rough focus knob till the

material on the slide comes in to focus.

9. Turn the fine focus knob till the image becomes sharp. The specimen should now

be sharply focused but may be unevenly illuminated.

10. Look down the eyepiece and move the mirror till the field of view becomes

brightly and evenly filled with light. The specimen should now be in sharp focus

and evenly illuminated but may be too bright.

11. Remove the eyepiece, look down the body tube and adjust the iris till the disc of

light takes up ¾ of the circle.

12. Replace the eyepiece. The microscope should now be set correctly under x10

magnification.

13. Manoeuvre the slide until the desired part of the specimen is in view.


14. Adjust the fine focus if necessary.

15. If appropriate, make a labelled diagram in your notebook/diary. Include a heading

and magnification.

16. Rotate the turret to bring the x40 objective lens above the slide.

DO NOT ADJUST THE ROUGH FOCUS KNOB

17. Remove one eyepiece, look down the body tube and adjust the iris till the disc of

light takes up ¾ of the circle. Replace the eyepiece.

18. Using only the fine focus knob make the image sharp.

19. Record briefly in your lab diary/notebook the procedure you have carried out and

make drawings of the specimen. Make sure your diagrams have headings and that

the magnification is included.

The following applies only if you are using x100 objective.

20. Turn the turret till the x 40 and x 100 lenses are at equal distances from the slide.

21. Carefully place a single drop of immersion oil on the slide. If immersion oil

drips on to any other part of the microscope, wipe it off immediately and

inform your teacher/lecturer or the technician.

22. Rotate the turret to place the x100 objective lens in the oil on the slide.

23. DO NOT ADJUST THE ROUGH FOCUS KNOB

24. Remove one eyepiece, look down the eyepiece tube and adjust the iris till the disc

of light takes up ¾ of the circle. Replace the eyepiece.

25. Using the fine focus control make the image sharp. Make drawings of the

specimen.

26. When you have finished working with the microscope, clean the lenses thoroughly

as you have been shown before putting it away.


5.3: Microscopic examination of yeast, pond water and hay infusion

Water in a pond or stream contains many different types of micro-organisms. In thispractical, you will use a cavity slide which has a little pit in its centre to examine asuspension of yeast or pond water. The drop is suspended from a coverslip into thecavity to make a ‘hanging drop preparation’. This prevents the micro-organisms frombeing flattened and allows you to observe them moving around.

You may also examine a ‘hay infusion’. This is prepared by placing three or fourpieces of clean dry hay or lawn cuttings in a Petri dish and covering them with pondor stream water. The lid is then replaced and the dish is kept at room temperature forone to two weeks away from direct sunlight.


Activity 5.3.1: Cavity slide preparation

MaterialsMaterials required by each studentLab coatDisinfectant and paper towelsDiscard jar with disinfectantLens tissueCavity slide and coverslipVaselineCocktail sticksPasteur pipette (plugged) and bulbYeast suspension, pond water or hay infusion

Materials to be sharedBenchkote if necessary

InstructionsInstructions are for right handed people. If you are left handed, please reverseinstructions accordingly.1. Wear a lab coat.

2. Clean slides and coverslips using lens tissue.

3. Using a cocktail stick place a rim of vaseline round the edge of a coverslip.

4. Place a bulb on a Pasteur pipette.

5. Holding the pipette in the right hand, remove the lid from the stock culture with

the left hand and draw up some liquid into the pipette.

6. Place one drop on to the centre of the coverslip (Fig 13) and discard the pipette

into a jar of disinfectant.

7. Lower the cavity slide on to the coverslip and quickly invert the slide andcoverslip (Fig 13).

vaseline pondwater

coverslip invert slideslide

coverslip

Fig 13: To make a cavity slide preparation


5.3.2: Bright field microscopy

In bright field microscopy, most of the contrast (differences between the brightness ofthe field and its background) is produced by the specimen absorbing light. Much ofthe direct light is either not absorbed or misses the specimen altogether. The resultingfield of view therefore appears fairly brightly lit.

Activity 5.3.2: Examination of a cavity slide preparation using bright fieldmicroscopy

Materials (5.3 .2, 5.3.3, 5.3.4)Materials required by each studentLab coatDisinfectant and paper towelsDiscard jar with disinfectantMicroscopeCavity slide (hanging drop) preparation


Instructions1. Using the instructions for setting up the microscope given earlier, examine the

prepared hanging drop under x10 objective (it helps to focus on the edge of the

drop first) and then under x40 objective.

2. In your lab diary/notebook record briefly the procedure and note whether the

sample contains one or many types of micro-organisms. If there is a variety,

record in a table the numbers of algae, protozoa and bacteria that you observe and

which type of micro-organism is most common in your sample.

3. Keep your slide preparation to view under dark field and phase contrast

illumination.

4. When finished with slide and coverslip, dispose into discard jar.


5.3.3: Dark field microscopy

In the previous practical, you will have noticed that many micro-organisms are almostcolourless and it is very difficult to observe them using bright field microscopy. Darkfield illumination is a technique by which the organisms appear brightly illuminatedon a dark background. A special condenser is used which prevents light rays fromentering the objective lens unless they have bounced off organisms on the slide.

You can get some idea of what dark field microscopy is like by closing the irisdiaphragm and illuminating the specimen from above.

Activity 5.3.3: Examination of a cavity slide preparation using dark fieldmicroscopy

Instructions

1. View the slides you have prepared for the previous practical under dark groundillumination.

2. In your lab diary/notebook, record the differences you observe in the field of view(the circular area you observe when looking down the eyepiece) and which type oforganisms you can see more clearly.


5.3.4: Phase Contrast Microscopy

It is difficult for a human eye to distinguish details inside cells because light intensitychanges only slightly when light passes through living cells. The technique of phasecontrast microscopy makes a living cell appear to have a different intensity to itsbackground. The observer can then distinguish cell contents more clearly.

Activity 5.3.4: Examination of a cavity slide preparation using phase contrastmicroscopy

Instructions (if a phase contrast microscope is available).1. View the slides you have prepared for the previous practical under phase contrast

microscope.

2. In your lab diary/notebook, record the differences you observe in the field of viewand the organisms.


5.4: Calculation of specimen size using a microscope

Specimens examined under the microscope are generally too small to be seen with thenaked eye. They are measured in micrometres. Viruses may be measured innanometres (nm). In general, viruses are too small to be seen with the lightmicroscope.

1µm = 10-3 millimetres (one thousandth of a millimetre)= 10-6metres (one millionth of a metre)

1nm = 10-6 millimetres (one millionth of a millimeter)= 10-9 metres (one thousand millionth of a metre).

The following table indicates the sizes of different types of cells.

Table: Sizes of micro-organisms

Size Units

1mm 10–3m

100µm 10–4m

10µm 10–5m

1µm 10–6m

100nm 10-7m

10nm 10-8m

Organism

Algae Protozoa Fungi

Bacteria

Viruses


Measuring objects using a microscope

Before you can measure the dimensions of a micro-organism, you must determine thesize of the microscope field of view (the circular area observed when you look downthe eyepiece). To accomplish this, you must use a special slide on which is printed amillimetre scale graduated in hundredths of a millimetre. This is called a stagemicrometer. A stage micrometer (Fig.14) is a very small ruler with each divisionequal to 0.01mm (10µm).

Fig. 14: Stage micrometer (micrometer slide)

It is not possible, however, to line up specimens on a slide next to this.

To measure the sizes of specimens accurately, you must use an eyepiece micrometer(a small disc of glass marked with a scale which is placed in an eyepiece). When thisis in place, you can see its graduations and the slide on the stage in the same field ofview (Fig.15). You can line up the object being observed alongside the graduationson the eyepiece micrometer and measure the length. (Fig.16).

Fig. 15: Eyepiece micrometer


Fig. 16: Measuring cells against eyepiece micrometer

Graduations on the eyepiece are arbitrary (i.e. they have no units) and the actuallength represented by each one will vary with the objective being used. For eachobjective, the eyepiece micrometer must be calibrated against the distance measuredon a stage micrometer (Fig.17).

The eyepiece micrometer is calibrated for each objective by superimposing its scaleon that of the stage micrometer. The number of stage micrometer divisions that areequivalent to a number of eyepiece divisions is noted and the following calculationcarried out.

One eyepiece unit = number of stage micrometer divisions x 0.01 µmNumber of eyepiece divisions


Fig. 17: Eyepiece micrometer over stage micrometer under different objectives


Questions: Calculation of specimen size using a microscope

1. Give the symbols for the following units: metre, millimetre, micrometre.

2. How many millimetres are in 1 metre?

3. How many micrometres are in 1 metre?

4. How many micrometres are in 1 millimetre?

5. Copy and complete the following table.

Metres millimetres micrometres

3 3000 (3x103) 3000000 (3x106)

0.5

70

425

0.8

39

0.59

0.02

6

26


Activity 5.4.1: Calculation of specimen size using a microscope

Materials requiredMaterials required by each studentMicroscope set up with eyepiece micrometerStage micrometer slidePrepared slides of micro-organismsLens tissue



Instructions

Calibration of eyepiece micrometer1. Set up microscope on low power with the stage micrometer in focus.2. Looking down the eyepiece, superimpose the eyepiece micrometer graduations on

the stage micrometer scale (Fig. 17).3. Adjust until the ‘zero’ marks on each scale are superimposed.4. Count the number of eyepiece divisions (x) which are equal to a number of

graduations (y) on the stage micrometer. If you have any doubt which scale iswhich, rotate the eyepiece and you will see that scale turning. Each stagemicrometer graduation equals 10µm. In your lab notebook/diary, briefly recordwhat you have done. Carry out the following calculation using your own valuesfor x and y.

Calculationx eyepiece units or divisions (epu) = y micrometer units

= y x 10µm1 epu = y/x x 10µm

Example x4 objective(see Fig. 17a)

32 eyepiece units (epu) = 100 micrometer units1 epu = 100/32 x 10µm1 epu = 31.3µm

5 Repeat the above for the high power objective.6 Carry out the calculation to calibrate the eyepiece micrometer at high power in

your notebook/diary.

Measurement of specimens7 Remove the stage micrometer.8 Using low power, focus a slide carrying the organism to be measured.9 Measure the length of the organism against the eyepiece micrometer graduations.10 Carry out the following calculation.

Calculation

Length of cell = a epu1epu (for x4) = 31.3µmLength of cell =

=a x 31.3 µm31.3a µm

11. Repeat for two other cell types at low power and also at high power.


Activity 5.4.2: Exercise in calculation of specimen size

Use the examples given in Activity 5.4.1 as a guide for these calculations.

a) Calibration of eyepiece micrometer.Calibrate the eyepiece micrometer for x10 and x40 objectives in Fig.17b and 17c.

b) Measurement of specimens

Fig. 18 shows Amoeba and Paramoecium cells as seen under the x10 objective andyeast cells under the x40 objective, with an eyepiece micrometer in place. Measurethe lengths of the cells against the eyepiece micrometer.

Using your answers and the values you have calculated in part a), work out thelengths of each type of cell.


Fig.18 (a): Amoeba cells x10 objective

Fig.18 (b): Paramecium cells x10 objective

Fig.18 (c): Yeast cells x40 objective

Fig.18: Measurement of microscopic specimens



6: STAINING

In general, the cytoplasm of protozoa, fungi and bacteria is almost colourless so it isdifficult to see them using an ordinary microscope. It is important to be able to seebacteria because the size, shape and grouping of the cells help to identify them.

To observe bacteria and yeasts better, the microbiologist first fixes them to a glassslide then stains them with a dark stain to provide a contrast to the bright background.Protozoa and fungal hyphae can be examined by staining the living organism.

FixationBacteria must be fixed to a slide to prevent them being washed off when stain isapplied. A smear of bacteria is made on a clean slide and dried thoroughly in air. It isthen heated gently in a flame. This kills vegetative cells, sticks them to the slide,makes them more permeable to stain and prevents them from breaking down. Themicrobiologist is therefore examining dead cells.

StainingA solution of dye is applied to the fixed smear.

Simple stains confer the same colour on all the cells which then appear a differentcolour to the background. Methylene blue stains cells blue. Carbol fuchsin andsafranin stain cells red.

Negative stains colour the background. The micro-organisms remain unstained andstand out brightly against a dark background. Common negative stains are India inkand nigrosin.

Vital stains are used to show up live cells. When irrigated with a vital stain like eosinor neutral red, cells such as Paramoecium and yeast will take up the stain byphagocytosis. When observed under the microscope for a period of time, thebackground at first appears red and the cells colourless. Gradually, the living cellstake up the dye with the result that live cells become red and the background becomespaler. The contrast of the cells is thus improved.


Activity 6.1: Preparation of a smear of bacteria or yeast from a solid culture

MaterialsMaterials required by each studentLab coatEye protectionDisinfectant and paper towelsDiscard jar with disinfectantLens tissue3 glass slidesLabelsPlate cultures of yeast and named bacteriaLoopForcepsBottle of sterile water (bijoux or universal)Bunsen burner and mat

Materials to be shared

Benchkote if necessary

Instructions (Fig.19)1. Wear a lab coat and use eye protection.

2. Collect materials.

3. Clean slide thoroughly using lens tissue. If necessary pass it through a hot Bunsen

flame to remove grease.

4. Label one end of the slide with initials, date and organism (use a self adhesive

label, wax pencil or glass marker).

5. Flame loop.

6. Using aseptic technique, transfer a loopful of sterile water on to the centre of the

slide.

7. Flame loop.

8. Using aseptic technique, transfer a very small part of a single colony from the

plate culture into the water and mix well, making sure that the smear is not too

thick.

9. Flame loop and place it on a heat resistant mat.

10. Using forceps to hold the slide and with the film downwards, pass the smear

through a yellow/blue flame several times to ‘fix’ it.

11. Place on a heat resistant mat to cool.

The smear is now ready to be stained.


Fig. 19: Preparation of a smear of bacteria or yeast



The purpose of simple staining is to show up the shape and arrangement of bacterialcells. The bacteria take up the stain and they appear coloured. You will use thesimple stain, methylene blue. Staining should be carried out on a rack placed over asink or draining dish.

Activity 6.2: Staining a smear preparation with a simple stain

MaterialsMaterials required by each student

Lab coatDisposable glovesDisinfectant and paper towelsDiscard jar with disinfectantFixed smears of Spirillum serpens, Bacillus subtilis and Micrococcus luteus and yeastMethylene blue stainBlotting paper (fibre free)Distilled water bottleStaining rack and draining dishForcepsMicroscopeImmersion oil


Instructions (Fig. 20)1. Wear a lab coat and eye protection.


3. Place previously fixed slides on staining rack over sink or draining dish.

4. Flood with methylene blue for 3 minutes.

5. Holding the slide at an angle with forceps over a sink or draining dish, wash wellwith distilled water.

6. Blot dry between two layers of fibre free blotting paper, taking care not to rub offthe cells.

7. Allow the slide to dry in air.

8. Examine under the microscope using the oil immersion lens if possible.Otherwise, use the highest magnification available.

9. In your lab diary/notebook, briefly record the procedure and draw diagrams of theorganisms you examined. Make sure that you record the name of the organismand the magnification used.

10. When finished, dispose of slides, gloves etc. as instructed.


Flood slide with stain

Wash slide with distilled water

Fig. 20: Staining a smear preparation with a simple stain




Title: Staining and MicroscopyName of candidate_____________________________________________________


a. Preparation for work is inaccordance with givenspecifications

• Hands washed




• Aseptic technique is used in preparation ofslide

• Slide stained according to instructions

• Microscope cleaned before use

• Microscope is set up correctly

• Slides are disposed of according to instructions

• Microscope is cleaned on completion of work.




• Heading

• Brief description of procedure including nameof micro-organism/sample and themagnification used.


• Description/labelled diagram of micro-organisms observed.

Organism:

Stain:


Date:


6.3: Observation of the filamentous fungi, Mucor and Penicillium usingLactophenol Blue

Lactophenol blue stains the fungal cytoplasm. The fungal walls appear colourlessagainst a light blue background. The fungal hyphae should be well teased out.

Activity 6.3: Staining a filamentous fungus using Lactophenol blue

MaterialsMaterials required by each studentLab coatDisposable glovesDisinfectant and paper towelsDiscard jar with disinfectantCovered beaker containing ethanolPlate cultures of Mucor and Penicillium2 slides and coverslipsLens tissueLactophenol blue (note: keep lactophenol off the skin)Forceps & mounted needleMicroscopeBunsen BurnerMat


Instructions (Fig. 21)1. Wear a lab coat and eye protection.2. Collect materials.3. Clean slide.4. Place a drop of lactophenol blue in middle of slide.5. Dip forceps in ethanol and replace lid.

6. Place forceps briefly in the Bunsen flame and allow ethanol to burn offcompletely.

7. Allow to cool.

8. Partially lift the lid of the Petri dish and use forceps to remove a small piece of thefungal colony

9. Replace lid.10. Place fungus in lactophenol blue and tease out well using forceps and needle.11. Flame forceps and needle with ethanol.

12. Using the mounted needle to support the coverslip, carefully lower it over thepreparation. Take care to avoid producing bubbles.

13. Examine under the microscope using the x40 objective lens.

14. In your lab diary/notebook, briefly record the procedure and make drawings of theorganisms you observe. Remember to note the name of the organism and themagnification used.


Fig. 21: Staining a filamentous fungus






e. Preparation for work is inaccordance with givenspecifications

• Hands washed



f. Techniques are carried outin accordance with safepractice and givenspecifications.







g. Record of work is clearand accurate.



• Heading




Organism:

Stain:


Date:



Nigrosin is a negative stain which colours the background. Yoghurt is used as asource of bacteria. The bacteria appear colourless on a blue-black background.

Activity 6.4: Observation of bacteria in yoghurt using a negative stain

MaterialsMaterials required by each studentLab coatDisposable glovesDisinfectant and paper towelsDiscard jar with disinfectantLive yoghurtSmall beakerNigrosin stainLens tissues3 glass slidesWire loopSterile water


Instructions (Fig. 22)

1. Wear a lab coat.2. Collect materials.3. Using aseptic technique, mix a little yoghurt with an equal quantity of distilled

water in a beaker.4. Flame loop.5. Place a loopful of the mixture at one end of a glass slide.6. Flame loop.7. Add one loopful of nigrosin stain and mix thoroughly using the wire loop.8. Flame loop.9. Prepare the film as described in Fig. 22. Do not fix with heat.10. When absolutely dry, examine under the highest magnification available. If

possible, use oil immersion.11. Note briefly in your lab diary/notebook the procedure you have carried out and

make drawings of the organisms you observe. Remember to title the diagramsand record the magnification.


1. Mix nigrosin with yoghurt

2. Place the end of slide B on the surface of slide A and pull it slowly towards thedrop of stain and yoghurt

3. The drop will then run along the edge of slide B

4. Push slide B quickly along slide A, dragging the drop behind – do this once only

Fig. 22: Negative staining of yoghurt






i. Preparation for work is inaccordance with givenspecifications

• Hands washed



j. Techniques are carried outin accordance with safepractice and givenspecifications.







k. Record of work is clearand accurate.



• Heading


l. Results and relevantobservations are reportedclearly.


Organism:

Stain:


Date:



Leguminous plants such as peas and clover form a symbiotic relationship with thebacteria, Rhizobium. Root nodules form when the bacteria are present (Fig. 23). Youwill examine a stained smear of the root nodules for Rhizobium. The bacteria appearas irregular X, Y, club, pear or star shaped bacteroids.

Fig.23: Rhizobium root system

Activity 6.5: Staining and observation of Rhizobium in root nodules

MaterialsMaterials required by each studentLab coatDisposable glovesDisinfectant and paper towelsDiscard jar with disinfectant3 glass slidesLens tissuesWire loopForcepsSterile distilled waterRoot nodules on legume rootsCrystal violet stainStaining rack and dish




Instructions1. Wear a lab coat and eye protection.2. Collect materials.3. Clean slides4. Using forceps, remove a medium sized clean nodule from the root and place in a

drop of water on the slide.5. Crush the nodule with a second slide and mash it in a drop of water.6. Using aseptic technique, make a smear of suspension on a fresh clean slide.7. When dry, flood with stain and leave for ten seconds.8. Wash with water.9. Allow to dry.10. Examine under the highest magnification available, if possible oil immersion.11. Note the procedure briefly in your lab diary/notebook and make drawings of the

organisms you observe. Remember to title the diagrams and note themagnification used.






m. Preparation for work is inaccordance with givenspecifications

• Hands washed



n. Techniques are carried outin accordance with safepractice and givenspecifications.







o. Record of work is clearand accurate.



• Heading


p. Results and relevantobservations are reportedclearly.


Organism:

Stain:


Date:


6.6: Vital staining

Vital stains are used to show up live cells. When irrigated with a vital stain such asneutral red, living yeast cells become red as they take up the stain. The backgroundbecomes paler as the stain enters the cells.

Activity 6.6: Staining of yeast with the vital stain, neutral red

MaterialsMaterials required by each studentLab coatDisposable glovesDisinfectant and paper towelsDiscard jar with disinfectantBunsen burner and matLens tissuesGlass slide and coverslipWire loopSterile waterPlate culture of yeastNeutral redBlotting paperMicroscope



Instructions1. Wear a lab coat and eye protection


3. Clean slide and coverslip.

4. Using aseptic technique, transfer two loopfuls of sterile water to the centre of theslide.

5. Using aseptic technique, transfer a small amount of yeast from a single colonyinto the water on the slide and mix.

6. Carefully lower the coverslip.7. Using the Pasteur pipette, draw up a little neutral red.

8. Slowly release the stain along one edge of the coverslip as shown by yourteacher/lecturer.

9. Place the edge of the blotting paper against the edge of the coverslip opposite todraw through the stain.

10. Observe under high power.

11. Record the colour of the background and the colour of the cells at five minuteintervals for a period of twenty minutes.





q. Preparation for work is inaccordance with givenspecifications

• Hands washed



r. Techniques are carried outin accordance with safepractice and givenspecifications.






• Microscope is cleared on completion of work.

s. Record of work is clearand accurate.



• Heading


t. Results and relevantobservations are reportedclearly.


Organism:

Stain:


Date:


Questions: Staining

1. Simple staining improves contrast. What does this mean?

2. Give two reasons for fixing a bacterial smear preparation.

3. Name two simple stains.

4. Explain what the term ‘negative stain’ means.

5. Name the type of stain which is taken up only by living organisms.


UNIT 2


TEACHER/LECTURER GUIDE


TEACHER/LECTURER GUIDE

ContentsBackground to unitMicrobiological procedures1: Preparation of work space2: Pouring plates3: Subculturing micro-organisms





5.1: Magnification













6.6: Vital staining of yeast with neutral redAnswers to questionsRecord of AttainmentChecklistsResources

Biotechnology: Working with Micro-organisms (Int 2) Teacher/lecturer guide 1

BACKGROUND TO UNIT

This unit of Intermediate 2 Biotechnology, ‘Working with Micro-organisms’ is apractical unit in which the student will learn some techniques of handling andstudying micro-organisms in the laboratory. These include plate pouring,subculturing micro-organisms, separating a mixed culture, staining and microscopy.

Since working with micro-organisms is covered by the COSHH regulations, it isnecessary to have suitable and sufficient control measures in place which have beendeveloped as a result of assessing the risks involved. An appropriate code of practiceon Safety in Microbiology can be adopted to meet these requirements.

To achieve this unit, the student must:• know and understand why microbiological, microscopy and staining techniques

are always carried out in a given way; and• become competent in performing a range of microbiological techniques and in

microscopy.

To achieve Outcome 1, the student must pass a written end of unit assessment whichwill test knowledge and understanding of the theory behind the practical techniques.The information needed for this is given in the Student Notes.

To achieve Outcome 2, the student must become competent in practicalmicrobiological techniques.

The time allowed for this unit is generous in order to allow the student to have ampleopportunity to practise the techniques a number of times until he/she becomes veryfamiliar with the procedures. The teacher/lecturer should teach students thetechniques included in the laboratory manual.

Each time the student performs a technique, he/she must• prepare for the work• carry out the techniques safely and according to instructions• keep a record of the work done• record results and observations clearly.

The student should keep a record of work and observations/results in a laboratorydiary/notebook and make an entry each time he/she carries out a piece of work orobserves results. The diary can be kept in a simple notebook or jotter.

Each entry should have:• the date when the work was carried out• a heading• a brief description of the technique the student carried out including incubation

times and temperatures• observations or results recorded in an appropriate manner (words, diagram, table

etc)• discussion of results if appropriate.


An example of a diary entry is given below.

Outcome 2 should only be awarded when the teacher/lecturer is satisfied that thestudent is competent in the techniques. It is not enough that he/she performs themsatisfactorily on only one occasion.

Checklists for Outcome 2 are provided in the Student Lab Manual and in this Guide.

Range of Outcome 2Each student must be competent in the following techniques:• Plate pouring• Subculturing micro-organisms. Students should be able to correctly remove

inoculum from solid medium (agar) and liquid medium (broth), and correctlyinoculate solid medium (agar) and liquid medium (broth). The Student LabManual includes instructions for a range of subculturing techniques (Activities3.1.1 – 3.1.4, 3.2.1 – 3.2.2, 3.3 and 3.4) which provide a range of alternativesthrough which this part of the Outcome can be achieved.

• Separating a mixed culture• Staining and microscopy. Activities 6.2, 6.3, 6.4, 6.5 and 6.6 provide alternatives

through which this part of the Outcome can be achieved.

Advice on Classroom Management for AssessmentSince students will be carrying out techniques several times, the class could bedivided so that different students are assessed for different alternatives on differentoccasions. It is not necessary to supervise a single student through a whole procedureunless the student is experiencing difficulty with the techniques. Rather, theteacher/lecturer could observe small groups of students unobtrusively.


Example of laboratory diary entry.

1.6.98 Subculturing, liquid to solid.Micrococcus luteus labeled ‘AA, M luteus, 27.5.98’, was asepticallytransferred from a broth culture to an agar plate and plated out to obtainsingle colonies using the streak plate method. The plate was incubatedupside down at 30°C for 72 hours and then placed in a refrigerator till itcould be examined.

The technique was also carried out with Saccharomyces cerevisiae but itwas incubated for five days at 30°C.

8.6.98 Results

Organism Single colonies Contamination

M luteus Yes No

S cerevisiae Yes Yes

Discussion A green fungus contaminated the S cerevisiae plate. Aspore from the air possibly entered the plate during theprocess of plating out and developed into the fungalcolony when the plate was incubated. The contaminatedplate cannot be used for further subculture as spores fromthe fungal colony may have contaminated the S cerevisiaecolonies.




Theory and practical work for students is contained the Student Lab Manual. TheStudent Summary Notes contain the information necessary for the Outcome 1Assessment. The following is additional information for teachers and lecturers.

Good working practice

The aim of this unit is for the student to acquire some of the basic technical skills usedin the study of microbes.

All organisms must always be treated as potential pathogens. Teachers and lecturersshould emphasise continually that students must follow safety procedures. If they donot, they cannot achieve the unit.

Students must be made aware of the principles of Safety in Microbiology through theStudent Lab Manual on Good working practice and the principles in a code ofpractice.

Safety is best guaranteed if routines are followed and become ‘second nature’ to thestudents. Teachers/lecturers must observe students continually with regard toensuring safety and in assessing each student's competency in the practical techniques.

Classroom Management

This applies to all practicals in this unit.Each should have his/her own set of equipment if possible and be able to sitcomfortably at the work bench with legs below the bench to protect them if there is aspillage.

Students should move around as little as possible and should work quietly.

The teacher/lecturer should demonstrate each technique. It may be advantageous toteach the method to small groups to ensure that each student can observe the processclearly.

Before starting each practical, students should be familiar with the underlyingtheory and understand the reasons for the safety control measures.




Type and purpose of activityThis activity can be used to:• develop knowledge and understanding of how to prepare the work space for

Outcome 1, PC a• illustrate good working practice• provide evidence for the assessment of Outcome 2:

PC a preparation of work is in accordance with given specificationsPC c the record of work is clear and accurate.

Background informationThe surface on which microbiological work is carried out must not be able to absorbany fluid or have cracks which could harbour micro-organisms. Some micro-organisms can survive for a considerable length of time in the environment and ifspilled on to a rough or absorbent surface could present a potential source ofcontamination both to others using the laboratory and to cultures. A sealed woodensurface is suitable. If the bench surface is unsuitable, it must be covered withBenchkote or some other non-absorbent material.

Whatever the surface, before starting work it must be swabbed with disinfectant toreduce the risk of contamination of cultures. The working area must be kept tidy anduncluttered in order to minimise the risk of accident. Allow only the essentials on thebench.

While carrying out the experiments in this unit, equipment will become contaminated.Contaminated items must be disposed of safely. There must always be a disposalcontainer within easy reach. If the student has to stretch, there is a risk of droppingthe apparatus or leaking organisms from a pipette for example.

A Bunsen burner is essential to the technique of aseptic transfer. It should be placedon the bench within easy reach of the operator. The Bunsen flame provides anupdraught which carries air away from the operator reducing the risk of breathing incontaminated air.

Classroom ManagementThe teacher/lecturer should demonstrate a well set up bench and emphasise theimportance of good bench management from both the safety angle and efficiency ofworking.

Students must prepare the workspace each time they carry out microbiologypractical. The teacher/lecturer should make students fully aware that they must dothis for each practical to achieve PC a (preparation for work is in accordance withgiven specifications).


ASSESSMENT/MARKING GUIDELINES

PC a: Teachers/lecturers should observe students following instructionssuccessfully and check that bench is set up as specified in theinstructions on each occasion they carry out practical work.

PC c: The note in the lab book is in accordance with student advice and isclear and accurate


2: POURING PLATES

Type and purpose of activityThis activity can be used to:• provide evidence for Outcome 2• develop knowledge and understanding of pouring agar plates for Outcome 1, PCa.

Background informationRefer to Student Notes.

Classroom ManagementThe teacher/lecturer should demonstrate the technique of pouring plates, if possible tosmall groups of students at a time.

It should be emphasised to students that if spilled, hot agar adheres to skin andcan cause a serious burn.

Students should be reminded that a checklist is included in the Student Lab Manualwhich can serve as a guide for achievement of this part of the range of Outcome 2.

ASSESSMENT/MARKING GUIDELINESTo achieve Outcome 2, students must complete this practical satisfactorily.

All students must therefore be made fully aware that they must record this activity andits results in a laboratory diary or notebook.

A checklist is included.




Title: Pouring platesName of candidate_____________________________________________________



• Hands washed







• Plates dried





• Heading







• Contamination.




Background informationSubculturing is the transfer of microorganisms from one medium to another. Itinvolves the aseptic removal of organisms from one culture medium followed byaseptic inoculation of another culture medium and is a technique essential to manybiotechnological procedures.

Students should be able to correctly remove inoculum from solid medium (agar) andliquid medium (broth), and correctly inoculate solid medium (agar) and liquidmedium (broth). The Student Lab Manual include instructions for a range ofsubculturing techniques (Activities 3.1.1 – 3.1.4, 3.2.1 – 3.2.2, 3.3 and 3.4) whichprovide a range of alternatives through which this part of the Outcome can beachieved.

The time allowed for this unit is generous because it is intended that students carry outthese procedures repeatedly and be given the opportunity to become competent inthem. They should be aware of the rationale behind each procedure and be consciousof safety considerations at all times.

Classroom Management for Activities 3.1.1 – 4.2 and 6.1 – 6.6The teacher/lecturer must stress the importance of safety procedures.

Students should develop a constant awareness of safety considerations andvehicles or entry points for contamination when working with micro-organisms.They must also understand and be encouraged to practise the importance of goodflaming technique.

The following should therefore be emphasised continually.• Inoculating loops or needles must be sterilised by flaming before and after use.

• Flaming must be carried out correctly.

• Once flamed, the loop must never be put down (or it could become contaminated)

but held in the hand and allowed to cool. Loops should not be waved around.

• The necks of tubes and bottles must be flamed after removing and before

replacing a lid.

• Caps of bottles must never be put down (to prevent contamination of or by them).

• Cultures should be exposed to the air for as short a time as possible.

• Lids of Petri dishes should be raised only enough to admit the wire loop and

permit the manipulation.

• Plates inoculated with swabs of the environment must be sealed.

• Certain sites must not be sampled.

It should be emphasised to students that any spillage must be reported to theteacher or lecturer in charge.


Incubation, refrigeration and disposalStudents must be given clear instructions regarding incubation, refrigeration anddisposal of materials. They must distinguish clearly between contaminated and non-contaminated waste and never dispose of contaminated material in a non-contaminated area. Consult an appropriate code of practice for further advice.

Activities 3.1.1 – 3.1.4: Loop transfer of micro-organisms.

Type and purpose of activityThis activity can be used to:• provide evidence for Outcome 2• develop knowledge and understanding of subculturing for Outcome 1, PC a• develop competence in the techniques of handling micro-organisms.

Background informationStudents will carry out solid to solid, solid to liquid, liquid to solid and liquid to liquidinoculations using a wire loop.

It is essential that they learn to hold and flame the wire loop correctly tominimise the risk of aerosol formation.

Background information regarding the rationale of the procedures is described fully inthe student support notes/background information.

Activities 3.2.1 and 3.2.2: Streak plate inoculations


The term ‘streaking out’ or ‘plating out’ is applied to the inoculation of solid mediain Petri dishes using a technique involving successive strokes with a wire loop. Theaim of this process is to obtain single colonies that can be further subcultured ifrequired. The loop is charged with a small amount of culture and several strokes inseries are then made on the surface of the medium with the aim of diluting thebacteria so that single cells are distributed on the surface. The loop is flamed betweeneach series of strokes to reduce the number of organisms and increase the chance ofisolating bacterial cells. On incubation, each single cell divides successively toproduce a single colony. In this way, pure cultures can be obtained.

The Petri dish should be turned through 90° between each series of strokes to allowthe loop to be held in a safe and comfortable position which remains the same foreach series of streaks. This is not shown well in the diagrams in the Student LabManual. It may help some students to draw the streaking pattern on the underside ofthe Petri dish with a fine tipped marker pen.




Background informationIt is sometimes difficult to pick up fungal mycelium with a wire loop or straight wire.Plate to plate inoculation is therefore often carried out using a sterile scalpel.

The scalpel may be sterilised by heating the tip in a Bunsen flame until red hot, aswith a wire loop. The blade may have to be held in the flame longer than a loop sothere is a risk that the handle may become hot.

Preferably, a scalpel wrapped in foil and sterilised by autoclaving may be provided.The student must dip the scalpel blade in alcohol, place it in the Bunsen flame,remove it from the flame and allow the alcohol to burn off and let the blade coolbefore use. This does not sterilise the blade since bacterial spores will resist thesetemperatures but avoids the problem of the scalpel handle becoming too hot to handle.

The scalpel is then used to cut through the fungal mycelium and agar below to extracta small block. This is then transferred aseptically to sterile agar and incubated.

Alternatively, transfer may be carried out using a cork borer flamed in ethanol or thewide end of a sterile Pasteur pipette. Here, a mounted needle flamed with ethanolmay be required to assist the transfer.

Young cultures (5 days) which are not sporulating heavily should be provided asinocula. Each inoculum should be taken from a non-sporing region possibly towardsthe edge of a colony to reduce the risk of contamination.



Background informationThe numbers and types of bacteria and fungi present on a surface such as thelaboratory bench or floor can be estimated by swabbing the area with a sterile swab orcotton wool bud which has been dampened with sterile water. The swab is thenrubbed across a sterile agar plate which is then incubated.


Plates must be sealed with tape and not opened after incubation since the organismsthat grow are unknown. Each Petri dish must be sealed diametrically usingtransparent adhesive tape and incubated base uppermost. Lids should not be sealed tobases around the circumference as this may create anaerobic conditions.

The relevant code of practice should be consulted regarding sites from which samplesshould not be taken.


From the range of activities, each student should demonstrate competence in• removal of inocula from solid media• removal of inocula from liquid media• inoculation of solid media• inoculation of liquid media.

All students must be made fully aware that they must record these activities and theirresults in a laboratory diary or notebook to achieve PCs c and d.

A checklist is included for guidance.






• Hands washed
















• Heading



Comments on:



• Contamination.




Type and purpose of activityThis activity can be used to:• provide evidence for Outcome 2• develop knowledge and understanding of subculturing for Outcome 1• develop competence in the techniques of handling micro-organisms.

Background informationIn this experiment, a mixed broth culture is streaked out on an agar plate thenincubated to obtain single colonies of different types of bacteria.

See Technical Guide for advice on organisms.

Classroom ManagementSee 3: ‘Subculturing micro-organisms’.










• Hands washed














• Heading



Comments on:






5: MICROSCOPIC EXAMINATION OF MICRO-ORGANISMS

Type and purpose of activityThis activity can be used to:• develop knowledge and understanding of the principles of the light microscope for

Outcome 1, PC b• develop competence in using a microscope.

Background informationThe microscope

For students to observe micro-organisms they must be able to obtain the best possibleperformance from the microscope.

The structure and function of the parts of the microscope are given in the Student LabManual.

In summary, the microscope consists of the following.A stand which bears:• a body tube which is raised and lowered with coarse/fine adjustment

mechanisms; sometimes the stage moves and the body tube is fixed; the nosepiececarries objectives.

• a stage on which slides to be examined are placed.• An optical system consisting of three parts.• A substage condenser fitted with an iris diaphragm. The condenser collects a

wide source of light from the mirror or electric bulb and condenses it to provide acone of rays of sufficiently wide angle to fill the objective being used. The irisdiaphragm controls the angle of the cone of light passing through the condenser.

• Objective lenses mounted on a rotating turret: low power lenses (x4, x10 or both),a high power lens (x40) and possibly an oil immersion lens (x100).

• The eyepiece or ocular lens at the upper end of the body tube. Magnification isusually x10 (x15 may be used in conjunction with x40 to view bacteria without oilimmersion).

Three focussing mechanisms :• a rack and pinion for adjusting the condenser assembly• a coarse adjustment for raising/lowering the body tube• a fine adjustment for raising/lowering the body tube.A light source. In school microscopes this is usually provided by a mirror tilted toreflect light through the objective lenses. Some microscopes may have a built-inlamp. In bench lamps, a 60 watt opal bulb should be used.



sharp cone.• The light passes through the opening in the stage into the slide holding the

specimen and may be absorbed, reflected or refracted by the specimen.• The light passes through the objective lens and forms a magnified image of the

specimen which is darker than the background (called the real image because itcan be projected on to a screen but is not seen by the microscopist).

• The image becomes an object for the eyepiece lens which magnifies the image asecond time to create a virtual image in space seen only by the observer.

5.1: Magnification

Type and purpose of activityThis activity can be used to:• develop knowledge and understanding of how magnification is calculated in a

light microscope for Outcome 1.

Background informationThe magnifying power is the degree of enlargement; that is, the number of times theimage appears greater than the original specimen.


Resolution

Resolving power or resolution is the ability to reveal closely adjacent structural detailsas separate and distinct. The resolving power of an optical microscope determines theamount of structural detail that can be observed and is limited by the wavelength ofvisible light.

The resolving power sets the limit of useful magnification which for a lightmicroscope is about x1500. Although lenses can be produced to produce muchgreater magnification than this, they will reveal no more detail and is described as‘empty magnification’. A good quality light microscope will resolve down to 2 µm.

DefinitionDefinition is the capacity of an objective to render the outline of the image of theobject clear and distinct. It depends on the elimination of optical aberrations inherentin the glass of the lenses.



Type and purpose of activityThis activity can be used to:• provide evidence for Outcome 2• develop knowledge and understanding of principles of the light microscope for

Outcome 1, PC b• develop competence in using a microscope.

Background informationStudents must become familiar with the structure of the microscope and becomecompetent at using it to view micro-organisms. The instructions provided in theStudent Lab Manual should allow them to do this. However, the followingadjustments will provide even better results and could be used if demonstrationmicroscopes are being set up for a class.

Precise adjustment of the condenser

The condenser is adjusted correctly when it is focusing an image of the lamp on thespecimen. To do this:1. Set up the microscope as far as instruction 12 in the Student Activity Guide.2. Place a pencil against the surface of the bulb (stand alone or integrated).3. Look down the microscope and move the pencil slowly over the lamp surface till

you can see it (possibly as a blurred image).4. Adjust the condenser till the pencil point comes into sharp focus. The condenser

is now focused correctly.

The condenser should be adjusted each time the microscope is set up and each timethe objective is changed.

Precise adjustment of iris

The Student Manual instructions include correct adjustment of the iris. It should beemphasised to the student that the iris must be adjusted each time the objective ischanged.

Condenser centring

Some student microscopes have centring condensers i.e. the condenser has to beadjusted so that the cone of light is in line with the objectives.

When the iris is closed down, the circle of light that can be seen should be centralwithin the body tube. If it is not, use the centring screws to centre the circle of light.When it is symmetric with the body tube, it is correctly positioned. There should beno need to readjust it although if handled roughly the condenser may be knocked offcentre.


Care of the microscopeDust from the air, grease from fingers, liquids and chemicals from slides all damagemicroscopes. Students should develop a routine of cleaning all the glass surfaces ofthe microscope with lens tissue before, during and after use. It should be stressed tothem that if immersion oil is left on lenses, it hardens and blurs the image. Ordinarytissue should not be used as it may scratch the glass surfaces of the lenses and leavefibres behind which obscure the image.

Only trained staff should carry out maintenance of the focusing mechanisms, oiling ofmoving parts and cleaning of the internal components of the lenses. SSERC canprovide technician training in maintaining and servicing microscopes.

Cleaning the objective

The front surface of an objective is likely to become marked by dust and fingerprintsand require regular cleaning. It can be cleaned as follows.1. Carefully unscrew the objective from the nosepiece and examine the front lens

(the one which is nearest the stage).2. Fold a clean sheet of lens tissue into a small pad and moisten it with xylol

substitute.3. Gently wipe the surface of the lens to remove dirt and grease. Take care that the

tissue is not too wet.4. Polish the surface of the lens with a clean sheet of lens tissue.Note: if immersion oil has dried on the lens, it may require several attempts to removeit.

Cleaning the eyepiece

The top lens of the eyepiece often becomes marked with dust and grease. It should becleaned in the same way as above. The other lens surfaces of an eyepiece should notneed cleaning.


Oil immersionOil immersion objectives are preferable to view bacteria satisfactorily because of theirgreater magnification and resolution properties. If they are not available, use a x15eyepiece with a x40 objective to give x600 magnification to observe bacterial shapes.

The x100 objective works very close to the object being viewed. Light rays passingthrough the glass slide to the air above are refracted so that many of them miss thefront lens of the objective, diminishing both the clarity and brightness of the image.

Immersion oil is used to reduce this refraction. Immersion oil has the same refractiveindex as optical glass. When oil fills the space between the slide and the objective,more light rays pass into the objective and the microscopist observes a muchimproved image.

Instructions for viewing a slide using oil immersion are provided in the StudentActivity Notes, ‘Setting up a microscope’.

Immersion oil is messy and it is important to stress to students that they must handle itcarefully and sparingly. They must not put it on lenses other than the oil immersionlens and if it is spilled on any other part of the microscope it should be wiped offimmediately with lens tissue. Oil immersion lenses should be cleaned with lens tissueimmediately after use.


Common problems and their solutions

Problem Cause SolutionImage is not sharp Dirty objective

Slide upside downClean with xylol substituteInvert slide

Dirty marksmarks move when slide ismovedmarks move when eye-piece is rotated in draw-tubemarks move whenobjective is rotated innosepieceMarks appear when lampis focused

Dirt or grease on slide

Dirt or grease on eyepiece

Dirt on objective

Dirt on lamp surface

Clean both sides with lenstissueClean top lens with xylolsubstitute and lens tissue

Clean front lens with lenstissue and xylol substitute

Clean lamp or defocuscondenser slightly

Too bright Iris too far openLamp too bright or tooclose

Close down iris slightlyChange bulb or move lampfurther away

Bright specimen on darkbackground

Light shining on top ofspecimen

Adjust position of lamp

‘Worms’ Fibres on slide, eyepiece orobjective

Clean with fibre free lenstissue

Dark edged circles Air bubbles Move slide and examinearea without air bubbles ormake fresh slide

Uneven illumination Mirror and/or lamp askewObjective not properlyclicked into positionFilter tray blocking lightinto condenser

Adjust mirror and /or lampRotate nosepiece

Swing in or out

Too dark Iris closed down too farLamp too weak or toodistantFilter tray obstructing lightinto condenserCondenser not focusedproperly

Open irisChange bulb or move lampcloserSwing in or out

Focus condenser

Image goes slowly out offocus

Insufficient friction infocus control mechanism

Adjust friction n focuscontrol (likely to requiretrained operator)

Jagged lines Coverslip edge or brokenpart of coverslip

Move slide to examinedifferent region. If c/slipbroken, make new slide


Classroom Management for Activities 5.2 – 6.6The teacher/lecturer should demonstrate how to set up a microscope and prepare eachtype of slide.

Students should be reminded to clean their microscopes after use.

Activities 5.3.1 – 5.3.4: microscopic examination of yeast, pond water and hayinfusion by bright field, dark field and phase contrast microscopy.

These would be useful as extension activities.

Type and purpose of activityThis activity can be used to:• provide evidence for Outcome 2• develop knowledge and understanding of principles of the light microscope and

magnification for Outcome 1, PC b• develop competence in using a microscope.

Activity 5.3.1: Cavity slide preparation

Background informationUse pond water from a non-contaminated pond or stream to make a hanging droppreparation. It should be rich in microscopic life and students should be able toobserve a variety of micro-organisms. Alternatively, a solution of yeast may be used.

Activity 5.3.2: Bright field microscopy

Background informationIn bright field microscopy, the specimen is illuminated directly from the light sourcepresenting a brightly lit field of view.

Activity 5.3.3: Dark field microscopy

Background informationDark ground illumination renders visible organisms which do not show up inunstained preparations under bright field microscopy.

Using a special condenser, the specimen is illuminated by oblique light only. Rays donot enter the objective unless scattered by objects e.g. cells which have a differentrefractive index from the medium in which they are suspended.

Alternatively, you can get some idea of what dark field microscopy is like by closingthe iris diaphragm and illuminating the specimen from above.

Organisms appear brightly illuminated on a dark background


Activity 5.3.4: Phase contrast microscopy

Background informationThe human eye can detect differences in amplitude of light waves but not differencesin phase.

Amplitude controls light intensity.

Using bright field microscopy it is difficult to observe unstained cells because lightintensity is similar before and after light passes through a living cell since theamplitude is not changed.

Phase oscillation of light waves, however, is changed by passage through a cell. Thehuman eye cannot detect differences in phase.

Essentially, the phase contrast microscope converts differences in phase to differencesin amplitude so that a living cell appears to have a different intensity to its backgroundand internal details of cells can be distinguished.


5.4: Calculation of specimen size using a microscope

Background informationSpecimen sizeThe specimens examined under the microscope are generally too small to be seen withthe naked eye. They are measured in micrometres (µm). Viruses may be measured innanometres (nm). In general, viruses are too small to be seen with the lightmicroscope.

1mm = 10-3metres (one thousandth of a metre)


1nm = 10-3micrometres (one thousandth of a micrometre)= 10-6 millimetres (one millionth of a millimeter)= 10-9 metres (one thousand millionth of a metre).



Size Units

1mm 10–3m

100µm 10–4m

10µm 10–5m

1µm 10–6m

100nm 10-7m

10nm 10-8m

Organism


Bacteria

Viruses



Type and purpose of activityThis activity can be used to:• provide evidence for Outcome 2.• develop knowledge and understanding of principles of the light microscope and

magnification and specimen size for Outcome 1, PC b• develop competence in using a microscope.

Background informationSee ‘Student Lab Manual’ for information on ‘measuring objects using a microscope’.

Activity 5.4.2 is a written exercise based on actual recorded measurements and can beused in addition to or as a substitute for Activity 5.4.1.

Activities 5.4.1 and 5.4.2 are fairly advanced activities and may be suitable asextension exercises for more able students. Description of an alternative practicalactivity is given below.

Alternative practical activitySimple micrometers printed on acetate may be purchase from standard suppliers andmounted on a glass slide.

Using this, a student can measure and record the diameter of the field of view underdifferent objectives.

By replacing the micrometer slide with a specimen, the student can estimate thelength of the specimen as a proportion of the diameter of the field of view. He/shecan then calculate an estimated length using the diameter previously recorded for thatobjective.


Activity 5.4.2: Exercise on calculation of specimen size

Type and purpose of activityThis activity can be used to:• develop knowledge and understanding of principles of the light microscope and

magnification and specimen size for Outcome 1, PC b

Background informationThis is a written exercise in which the student uses diagrams to calibrate an eyepiecemicrometer for different objectives and to measure different specimens.

This may be best suited to more able students.

Answers

Calibration of eyepiece micrometer

x 10 objective (Fig. 17b)

80 eyepiece units (epu) = 100 micrometer units

1 epu = 100/80 x 10µm

= 12.5µm

X40 objective (Fig. 17c)

100 eyepiece units (epu) = 30 micrometer units

1 epu = 30/100 x 10µm

1 epu = 3µm

Measurement of specimensAmoeba (Fig 18a) x10 objective

Length of Amoeba cell = 40 eyepiece units (epu)

1epu for 10 objective = 12.5µm

Length of cell =

=

40 x 12.5 µm

500 µm

Length of Amoeba cell = 500 µm


Measurement of specimens

Paramoecium (Fig 18b) x10 objective

Length of Paramoecium cell = 15 eyepiece units (epu)

1epu for 10 objective = 12.5µm

Length of cell =

=

15 x 12.5 µm

187.5 µm

Length of Paramoecium cell = 187.5 µm

Measurement of specimens

Yeast (Fig 18c) x40 objective

Length of yeast cell = 5 eyepiece units (epu)

1epu for 40 objective = 3 µm

Length of cell =

=

5 x 3 µm

15 µm

Length of yeast cell = 15 µm


6: STAINING

Background informationMost micro-organisms have a refractive index which differs only slightly from glassso it is difficult to observe them by bright field microscopy. The purpose of stainingis to improve contrast between the organisms and the bright background. Simplestaining confers the same colour on all cells. Differential staining (such as Gram’sstain) colours bacteria differentially. Students are required to carry out simplestaining only in this unit.

Simple stains are simple basic dyes e.g. methylene blue, methyl violet or basic fuchsinthat are applied in a watery solution to the cells. The coloured, positively chargedcation of the basic dye combines firmly with negatively charged groups in bacterialprotoplasm, particularly with phosphate groups. After application, the preparation iswashed to remove excess dye from the slide. The cells retain the dye and whenexamined under the microscope appear coloured in contrast to a bright background.

Acidic dyes have coloured anions and do not stain bacteria strongly (except at veryacid pH values). The technique of negative staining uses acidic dyes. Bacteria appearlight against a dark background.

Vital stains e.g. neutral red and eosin, are taken up by living cells by the process ofphagocytosis.

Classroom managementStudents should prepare for working with micro-organisms. The teacher/lecturershould emphasise that aseptic technique must always be used when manipulatingmicro-organisms.

The teacher/lecturer should demonstrate each technique.

Smears of a variety of organisms will be used for staining. Students may be providedwith prepared smears or make their own preparations. Students should receive clearinstructions regarding disposal of materials.

When working with stains, care must be taken to prevent hands from becomingstained. Well fitting gloves disposable gloves should be worn if available. A stainingrack (made from glass rods and rubber tubing (Fig.1) and dish per pair of studentsavoids crowding around sinks.

Fig. 1: A staining rack


Activity 6.1: Preparation of a fixed smear of bacteria or yeast from a solidculture

Background informationBefore making a smear, slides should be thoroughly cleaned using lens tissue.

Smears or films are made by aseptically mixing a little growth from a single colonywith a loopful of sterile water on a slide. It is important that only a minute quantity oforganism is placed on the slide and that the water/organism mixture is thoroughlyemulsified. The mixture is then spread evenly and thinly across the centre of theslide. Beginners tend to pick up too much material and thus make too thick asmear. Individual cells can only be distinguished in a thin film.

FixationBefore staining, bacteria must be fixed to the slide.

Fixing kills the vegetative cells, fixes them to the slide, makes them more permeableto the stain and prevents autolysis.

The film can be fixed by passing the dried slide, smear downwards, three timesthrough a blue/yellow flame or by heating the underside of the glass slide. Here, thesmear is uppermost and the slide held for a few seconds in the top of the Bunsenflame till the slide becomes hot. Fixation has taken place when the slide is just toohot to be borne on the back of the hand. Slides should be held with forceps and caremust be taken not to hold the slide too long in the flame. Eye protection should beworn as a precaution.

After cooling, the smears are ready to be stained.



Type and purpose of activityThis activity can be used to:• provide evidence for Outcome 2, PC a and b• develop knowledge and understanding of simple staining techniques for

Outcome 1, PC b.

Background informationWhen stained with methylene blue, bacteria appear blue against a bright background.The nuclei of yeast cells can often be observed as dark object in the cytoplasm. Cellwalls of both bacteria and yeast do not take up the stain and can often be noted ascolourless areas between cells.

The stain is poured directly on to the slide. After the due time, the dye is washed offwith water using a water bottle. The slide is held at an angle with forceps over thedraining dish and water directed on to it from the bottle.

The slide can then be dried between two sheets of fibre free blotting paper or left todry in air. Drying may be completed by heating gently over a Bunsen flame.

Bacteria should be viewed under oil immersion. Otherwise, use of a x15 eyepiecelens with a x40 objective lens should allow observation.

6.3: Observation of the filamentous fungi, Mucor and Penicillium usingLactophenol Blue

Type and purpose of activityThis activity can be used to:• provide evidence for Outcome 2, PC• develop knowledge and understanding of simple staining techniques for

Outcome 1, PC b.

Safety noteLactophenol blue is poisonous by absorption and should be kept off skin. Thefollowing control risk.• Good technique• Use of small volumes• Use of a dropping bottle• Use of a staining rack• Well fitting disposable gloves should be worn if available.

Background informationThe fungal cytoplasm takes up the blue stain. Colourless hyphal walls can beobserved against a light blue background. Fungal cultures should be 5 – 7 days old toallow observation of hyphae. Old cultures possess too many spores which mask thefilaments.


The fungal hyphae must be well teased out. When applying the coverslip, support itwith a mounted needle and lower it gently to avoid bubbles.

Remove excess stain with the edge of a piece of blotting paper placed against oneedge of the coverslip. Take care not to remove too much.

Activity 6.4: Negative staining of yoghurt with nigrosin

Type and purpose of activityThis activity can be used to:• provide evidence for Outcome 2• develop knowledge and understanding of simple staining techniques for

Outcome 1, PC b.

Background information‘Live’ yoghurt must be used. Bacteria can be seen as bright objects against a darkbackground.

Correct preparation of nigrosin is critical to success.

Activity 6.5: Staining of root nodules to observe Rhizobium

Type and purpose of activityThis activity can be used to:• provide evidence for Outcome 2• develop knowledge and understanding of simple staining techniques for

Outcome 1, PC b.

Background informationRhizobium can be observed from root nodule preparations as described in the studentguide.

Activity 6.6: Staining of yeast with the vital stain, neutral red.

This activity can be used to:• provide evidence for Outcome 2• develop knowledge and understanding of simple staining techniques for

Outcome 1, PC b.

Background informationUse a growing culture of yeast (or live protozoa). Only live cells take up the vitalstain. The cells at first appear colourless against a red background but as they take upthe stain by phagocytosis, living cells become red in contrast to the background whichpales in colour. This can be observed over a period of about twenty minutes. Neutralred turns yellow in acid condition. Food vacuoles can be observed as yellow.


ASSESSMENT/MARKING GUIDELINESTo achieve Outcome 2, students must successfully stain and observe micro-organismsby one of the methods in Activities 6.2, 6.3, 6.4, 6.5 or 6.6.








a. Preparation for work is inaccordance with givenspecifications

• Hands washed













• Heading





Answers to Questions

1.: Preparation of work space

1. Wash hands and put on a lab coat.2. A non-absorbent surface is required to ensure that micro-organisms cannot

penetrate the surface but can be removed easily.3. The surface is disinfected before and after use to reduce the number of potential

contaminants.4. All equipment should be within easy reach of the operator.5. Windows and doors should be kept closed to minimise draughts and reduce the risk

of contamination.6. The Bunsen burner provides an updraught which carries air upwards and away

from the operator reducing the risk of contamination.

2: Pouring plates1. Labels should be written on the base of plates.2. Three details that should be included in labels are: name, date and type of agar.3. Necks of bottles should be flamed before pouring agar.4. Bubbles on the surface of the agar can be avoided by ensuring it is cooled to the

appropriate temperature , not agitating it and pouring and swirling it gently.5. Excess condensation on lids can be prevented by ensuring that the temperature of

the agar is low when poured and by drying the plates.6. Plates should be stored upside down.7. Plates should have a smooth surface, even layer of agar, the base of the dish

should be covered and there should be no contamination.

3.1: Loop transfer of micro-organisms1. A wire loop should be held loosely in the hand in a similar way to holding a pencil.

It should be held almost vertically to prevent backflow of liquid and should neverbe waved around.

2. The pale blue cone of the Bunsen flame is at a lower temperature than the rest ofthe flame. This allows any liquid on the loop to evaporate without spurting off theloop and prevents formation of aerosols containing live organisms.

3. The loop is sterilised when it has become red hot.4. The loop must be cooled before subculturing to avoid killing the organisms.5. a) necks of bottles should always be flamed and b) lids should never be put down

on bench.6. The loop must be flamed at the end of every subculturing procedure.7. Lids could be transferred inadvertently.8. Cultures should always be labelled with name or initials, date and organism.9. Condensation is thus prevented from dropping on to the surface of the agar plates.10. Cultures must be sterilised before final disposal to ensure that they do not pose a

risk of contamination.


3.2: Streak plate inoculation

1. The streaking out technique is used to obtain single colonies.2. The loop is flamed between strokes to reduce the number of micro-organisms on

the loop.3. A pure culture contains only one type of organism.4. A colony is produced from a single cell which divides repeatedly.

3.3: Plate to plate transfer of fungal mycelium1. Fungal mycelium is the hyphal growth a fungus produces giving it a hairy

appearance.2. It is more difficult to pick up the fungal mycelium with loops and needles.3. Fungal mycelium is not streaked across the plate but inoculated in a central

position to allow the colony to grow outwards.

3.4: Use of sterile swabs to sample the environment

1. Swabs are made of cotton wool and would burn in a Bunsen flame.2. Make sure you do not touch the swab end. Only touch the handle.3. The organisms which grow are unknown and may be dangerous.

4: Separating micro-organisms

1. A mixed culture contains more than one type of organism.2. Streaking or plating out will dilute and separate micro-organisms until single cells

are deposited on the plate.3. Streak out to obtain isolated single colonies; use an isolated single colony as the

inoculum for further streaking out.

5: Structure and function of the light microscope

1. The purpose of the light microscope is to view organisms or structures too smallto be seen with the naked eye.

2. The iris controls the angle of the cone of light which passes through thecondenser.

3. The stage supports the slide.4. Three parts of the microscope which contain lenses are the eyepiece, the

objectives and the condenser.5. The focus controls are adjusted to make the image sharp.

5.1: Magnification

Eyepiece lensmagnification

Objective lensmagnification

Totalmagnification

x15 x10 x150x10 x10 X100

X10 x40 x400x10 X100 x1000x15 x40 X600


5.4: Measuring objects using a microscope

1. Metre = m; millimetre = mm; micrometre = µm.2. There are one thousand millimetres in a metre.3. There are one million micrometres in a metre.4. There are one thousand micrometres in a millimetre.5.

Metres millimetres micrometres3 3000 (3x103) 3000000 (3x106)

0.5 500 (5x102) 500,000 (5x105)0.07 (7x10-2) 70 70,000 (7x104)

0.000425 (4.25x10-4) 0.425 (4.25x10-1) 4250.0008 (8x10-4) 0.8 800 (8x102)

0.000039 (3.9x10-5) 0.039 (3.9x10-2) 390.00059 (5.9x10-4) 0.59 590 (5.9x102)

0.02 20 20,000 (2x104)0.000006 (6x10-6) 0.006 (6x10-3) 60.026 (2.6x10-2) 26 26,000 (2.6x104)

6: Staining1. Improving the contrast means that there is a greater differentiation between cells

and background.2. Fixing a smear attaches the cells to the slide, kills the cells and makes them more

permeable to stain.3. Methylene blue, crystal violet.4. A negative stain does not stain the cells. They appear bright on a dark

background.5. A vital stain.


Unit 2:Working with Micro-organisms

Record of Attainment Class YearOutcome 2

subculturingCandidate Outcome 1score From

solidFromliquid

To solid Toliquid

Separatinga mixedculture

Stainingand

microscopy

O2achieved

Unitachieved



Unit 2: Working with Micro-organisms (Int 2)Title: Pouring platesName of candidate_____________________________________________________



• Hands washed







• Plates dried





• Heading







• Contamination.







• Hands washed
















• Heading



Comments on:



• Contamination.







• Hands washed














• Heading



Comments on:









e. Preparation for work is inaccordance with givenspecifications

• Hands washed



f. Techniques are carried outin accordance with safepractice and givenspecifications.







g. Record of work is clearand accurate.



• Heading





RESOURCES

Videos‘Aseptic Techniques’: illustrates principles and techniques necessary for asepticmicrobiology work. There are demonstrations in a university laboratory setting andthree industrial settings, the Courage Brewery, Roche Products Ltd and the PublicHealth Laboratories.

‘An Introduction to Microscopy’: covers the basic optical properties of themicroscope and demonstrates the correct procedure to set up bright field illumination.It shows the technique of oil immersion and describes the calibration of the eyepiecegraticule.

Available from Shotlist, The EBS Trust, 36-38 Mortimer Street, London W1N 7RB.Tel: 0171 765 4635; Fax: 0171 580 6246; e-mail: [email protected]

Web sitesNational Centre for Biotechnology Education: http://www.ncbe.reading.ac.uk‘The microbial world’, a resource compiled by Jim Deacon at Edinburgh Universitywhich covers bacteria, fungi and viruses:http://helios.bto.ed.ac.uk/bto/microbes/microbes.htm.

Useful addressesSSERC (Scottish Schools Equipment Research Centre)St Mary’s Land, 23 Holyrood Road, Edinburgh EH8 8AETel: 0131 558 8180; Fax 0131 558 8191; e-mail: [email protected].

Science and Plants for Schools (SAPS) Biotechnology Scotland, Room 7.05,Darwin Building, King’s Buildings, University of Edinburgh, EH9 3JRTel: 0131 650 7124; Fax: 0131 650 8650

CCAP (Culture Collection of Algae and Protozoa)Institute of Freshwater Ecology, Windermere Laboratory, Far Sawrey, Ambleside,Cumbria LA22 0LPTel: 01539 442468; Fax: 01539 446914; e-mail: [email protected] Bio, Portland Press Ltd, Commerce Way, Colchester CO2 8HPTel: 01206 796351

BBSRC Science Club, Polaris House, North Star Avenue, Swindon SN2 1UHTel: 01793 413200 or 413302


UNIT 2


STUDENT SUMMARY NOTES


STUDENT SUMMARY NOTES

ContentsMicrobiological procedures1: Preparation of work space2: Pouring plates3: Subculturing micro-organisms4: Separating micro-organisms5: Microscopic examination of micro-organisms

Magnification

Specimen size6: Staining

Biotechnology: Working with Micro-organisms (Int 2) Student summary notes 1


All organisms must always be treated as if they could cause disease so recommendedsafety procedures must be followed.

The aims of these safety procedures are:• to avoid bringing contaminating organisms to the laboratory bench• to avoid contaminating laboratory cultures• to avoid contaminating yourself, your colleagues and your surroundings• to avoid taking contaminating organisms out of the laboratory.

Good Working Practice

To achieve the above aims, the following good working practice must be observed.

General safety procedures in the laboratory

• Do not eat, drink or smoke.

• Do not lick fingers or labels.

• Avoid touching the face.

• Tie back long hair.

• Cover any cuts and grazes with a waterproof plaster.

• Wear a lab coat and keep it fastened.

• Speak quietly and avoid unnecessary movement around the laboratory.

• Report all accidents, no matter how trivial, to the teacher/lecturer.

• Do not pipette by mouth.

• Keep doors and windows closed.


On entering the microbiology laboratory

9. Hang up outdoor coats.

10. Leave bags at the side of the room taking only essentials to the workbench.


12. Dry hands using disposable paper towels.

13. Cover any wounds with a waterproof plaster.

14. Put on a lab coat.

15. Collect disinfectant and paper towel.


17. Do not touch your bag, outdoor wear etc. without first washing your hands.

18. Do not leave the laboratory wearing a lab coat.

Bench Management

7. Swab bench with disinfectant at start and end of work using a paper towel.

8. Sit on the lab stool with legs under the bench.

9. Organise the equipment you are using so that it is within easy reach.

10. Keep bench as uncluttered as possible.

11. Do not lay contaminated materials on bench.

12. Report all spillages and breakage to the teacher or lecturer in charge.

Before leaving the microbiology laboratory


2. Place stool under bench.

3. Remove and put away lab coat.


5. Collect outdoor coat and leave laboratory.



Preparing yourself

Before starting work you must wash your hands thoroughly with soap and water. Youshould then put on a lab coat to protect your own clothes.

Preparing the work space

The surface on which you work must be smooth and non absorbent to ensure it cannotharbour micro-organisms. If the bench surface is unsuitable, it should be coveredwith Benchkote or other non absorbent material.

Before starting work you must swab your work space with disinfectant to reduce therisk of contamination of your cultures.

You should always have a disposal container of disinfectant within easy reach.

A Bunsen burner is essential to the technique of aseptic transfer. The Bunsen flameprovides an updraught which carries air upwards away from the operator reducing therisk of contamination.

Air movement should be kept to a minimum in microbiology laboratories to preventcontamination. While practical work is being performed, doors and windows shouldbe kept shut.

In professional microbiology laboratories, specialised cabinets are used for thetransfer of micro-organisms for safety reasons. Transfer chambers are such cabinets.The operator works with their hands and forearms in the chamber, transferringmicroorganisms, preparing sterile media etc. Lamina flow chambers or cabinets aremore sophisticated types of transfer chamber.



2: POURING PLATES

Agar

Liquid media can be made solid by the addition of agar.

The melting and solidifying points of agar are not the same. At the concentrationsnormally used, most agars melt at about 95°C when heated but solidify only whencooled to about 42°C.

Pouring Agar Plates

Agar plates are prepared by pouring liquid agar at 55°C into sterile Petri dishes andallowing it to solidify.

Before pouring, unopened plates are labelled on the underside using an indelible penor wax pencil with initials, date and type of agar. This prevents confusion should lidsinadvertently be swapped. The plates are then placed the right way up ready toreceive molten agar.

Sterile nutrient agar in universal bottles or other containers is first melted by heatingto 100°C then cooled in a water bath to 55°C. Using aseptic technique, a bottle neckis flamed and the agar poured gently into a Petri dish on a flat surface, raising the lidof the dish only far enough for the mouth of the bottle to enter. The lid is thenreplaced and the plate left undisturbed until the agar has cooled and set.

It is essential that the surface of the medium should be dry in order to maintain singlecolonies. Condensation produced from the cooling of the agar can make the agarsurface wet but is normally reduced by pouring the agar at 55°C and if necessary theplates can be dried open and upside down in an undisturbed area.

Plates are always incubated and stored in the inverted position to preventcondensation dropping on to the agar surface.




Subculturing is the aseptic transfer of micro-organisms from a culture to freshmedium.

The use of aseptic technique minimises the risk of contamination of cultures and alsoreduces the risk of micro-organisms from the laboratory cultures escaping to theenvironment.

Flame sterilisation of instruments

Metal instruments used to transfer micro-organisms are sterilised using red heat in aBunsen burner before and after use. They must be heated till red hot to make sure thatany contaminating bacterial spores are destroyed.

Good flaming technique is very important to avoid contamination of the surroundingair with aerosols.

A Bunsen burner is lit and the air hole opened fully to provide a blue flame. Theoperator holds the loop between thumb and fingers as if holding a pencil very loosely,at an angle that is almost vertical.

Fig. 1: Holding a wire loopFor copyright reasons, the image is unavailable.


The loop is placed in the light blue cone of the flame. Positioning the loop in this coolarea of the flame allows any liquid to dry out and prevents formation of aerosols.Aerosols are fine liquid or solid particles that are dispersed into the air and mightcontain micro-organisms.

Fig. 2: Placing a loop in blue cone of flameFor copyright reasons, the image is unavailable.

After any liquid material has evaporated, the loop is drawn slowly up into the hottestregion of the flame (immediately above the light blue cone and held there until it isred hot.

Fig. 3: Drawing loop into hottest region of flameFor copyright reasons, the image is unavailable.


The loop is then withdrawn from the flame and allowed to cool before touchingmicroorganisms.

Fig. 4: Cooling the loopFor copyright reasons, the image is unavailable.

Straight wires may be sterilised in the same way.

Aseptic transfer operations

Once the loop has been sterilised and cooled, it is used to remove organisms (theinoculum) from a culture and inoculate sterile growth medium. When working withmicro-organisms, aseptic techniques are used to avoid escape of organisms to thesurroundings and contamination of the culture by micro-organisms from theenvironment.

To minimise the chances of contamination, cultures and media are exposed to the airfor the minimum time it takes to perform a manipulation or to make observations. Allsubculturing procedures are carried out close to a Bunsen flame.

After flaming, the loop is not put down until the procedure has been completed.

Lids of Petri dishes are never completely removed. They are opened just enough toallow entry of the loop to perform the manipulation and minimise exposure to the air.

Liquid cultures are disturbed as little as possible to reduce the risk of aerosolformation. Lids from cultures are never placed on the bench surface wherecontamination might occur. They are removed from the bottle or tube using the littlefinger, held there while manipulation of the culture takes place and then replaced.The rest of the hand is free to carry out the manipulation. Lids are tightened beforeincubation to prevent spillage.


To remove any contaminating organisms on the neck of a bottle, it is passed through ahot Bunsen flame on removal and before replacement of the lid.

The loop must be flamed to red heat when the subculturing procedure is finished.

The table below summarises potential points of contamination and the techniquesemployed to minimise the risk.

Contamination risk Precaution

Inoculating loop Flame and cool.Do not lay down loop until procedure iscomplete.Work close to Bunsen flame.

Opening of Petri dish (solid medium) Open lid for as short a time as possible.Open lid just enough to insert wire loop.

Opening of liquid cultures (bottles, tubes) Hold the lid in crook of little finger –never place on work bench.Pass neck passed through a hot Bunsenflame before insertion and afterwithdrawal of loop to kill anycontaminating organisms.



When professional microbiologists isolate micro-organisms from the environment oran infected person, it is extremely rare to obtain a pure culture. It is thereforenecessary to separate micro-organisms. Plating or streaking can be used to achievethis. Using isolated single colonies as inocula for further streak plates, pure culturescan be obtained.

The term ‘streaking out’ or ‘plating out’ is applied to the inoculation of solid media inPetri dishes using a technique involving successive strokes with a wire loop. The aimof the procedure is to obtain isolated single colonies. The loop is charged with a smallamount of culture and several strokes in series are then made on the surface of themedium with the aim of diluting the bacteria so that single cells are distributed on thesurface. The loop is flamed between strokes to reduce the number of organisms andincrease the chance of isolating single cells. On incubation, each single cell dividessuccessively to produce a single colony (Fig. 5). In this way, pure cultures can beobtained.

Fig. 5: Streak plate showing single colonies



5: MICROSCOPIC EXAMINATION OF MICRO-ORGANISMS

Micro-organisms are so small that they you cannot see them without the aid of amicroscope. Microscopes are used to produce an enlarged image of objects too smallto be seen with the naked eye.

Although microscopes produced by different manufacturers may look quite unlikeeach other, they all work on the same principle and consist essentially of similarworking parts.


sharp cone.• The light passes through the opening in the stage into the slide illuminating the

specimen.• The light passes through the objective lens and forms a magnified image of the

specimen which is darker than the background.• The eyepiece lens magnifies this image further and creates the image that the user

sees.

Magnification

The magnifying power is the degree of enlargement; that is, the number of times theimage appears greater than the original specimen.



Specimen size

Specimens examined under the microscope are generally too small to be seen with thenaked eye. They are measured in micrometres. Viruses may be measured innanometres (nm). In general, viruses are too small to be seen with the lightmicroscope.


1nm = 10-6 millimetres (one millionth of a millimeter)= 10-9 metres (one thousand millionth of a metre).



Size Units

1mm 10–3m

100µm 10–4m

10µm 10–5m

1µm 10–6m

100nm 10-7m

10nm 10-8m

Organism


Bacteria

Viruses

When observing an object under the microscope, it appears larger the greater themagnification.


6: STAINING

In general, the cytoplasm of protozoa, fungi and bacteria is almost colourless so it isdifficult to observe such cells using an ordinary microscope. It is important to be ableto see bacteria because the size, shape and grouping of the cells help to identify them.

To observe bacteria and yeasts better, the microbiologist first fixes them to a glassslide then stains them with a dark stain to provide a contrast to the bright background.Protozoa and fungal hyphae can be examined by staining the living organism.

FixationBacteria must be fixed to a slide to prevent them being washed off when stain isapplied. A smear of bacteria is made on a clean slide and dried thoroughly in air. It isthen heated gently in a flame. This kills vegetative cells, sticks them to the slide,makes them more permeable to stain and prevents them from breaking down. Themicrobiologist is therefore examining dead cells.

StainingA solution of dye is applied to the fixed smear.

Simple stains confer the same colour on all the cells which then appear a differentcolour to the background. Methylene blue stains cells blue. Carbol fuchsin andsafranin stain cells red.

Negative stains colour the background. The micro-organisms remain unstained andstand out brightly against a dark background. Common negative stains are India inkand nigrosin.

Vital stains are used to show up live cells. Paramecium and yeast, for example, willtake up eosin by phagocytosis. When observed under the microscope for a period oftime, the background at first appears red and the cells colourless. Gradually, theliving cells take up the dye with the result that live cells become red and thebackground becomes paler. The contrast of the cells is thus improved.



UNIT 2


TECHNICAL GUIDE


TECHNICAL GUIDE FOR LABORATORY MANUAL

ContentsBackground to unit1: Preparation of work space2: Pouring plates3: Subculturing micro-organisms

3.1: Loop transfer of micro-organismssolid to solid


3.1.3 solid to liquid

3.1.4 liquid to liquid


3.2.1. solid to solid
















6.6: Vital staining of yeast with neutral red

Biotechnology: Working with Micro-organisms (Int 2) Technical guide 1

BACKGROUND TO UNITThis unit of Intermediate 2 Biotechnology, ‘Working with Micro-organisms’ is apractical unit in which the student will learn some techniques of handling andstudying micro-organisms in the laboratory. These include plate pouring,subculturing micro-organisms, separating a mixed culture, staining and microscopy.

Since working with micro-organisms is covered by the COSHH regulations, it isnecessary to have suitable and sufficient control measures in place which have beendeveloped as a result of assessing the risks involved. An appropriate code of practiceon Safety in Microbiology can be adopted to meet these requirements.




Materials requiredMaterials required by each student:Benchkote and non-absorbent tape if required.Disinfectant and paper towelDiscard jar with freshly diluted clear phenolic disinfectantBunsen burnerWire loopCultures and media

Benchkote need only be used if laboratory work surfaces are absorbent and thereforeunsuitable for microbiological practical work. Depending on the numbers of studentsinvolved, the benchkote can either be cut to size of an individual work space or be cutto cover several work spaces. Alternatively, individual boards of kitchen worksurface type material could be used.

Advice on disinfectantsFor swabbing benches:• freshly diluted ampholytic surfactants (e.g. Gerrard ‘ASAB’/Harris ‘BAS’ or

similar) or 1% hypochlorite solutions (a good quality commercial bleach e.g.Domestos or laboratory sodium hypochlorite) available in bottles, sprays and/orbeakers. Use with paper towels for swabbing.

For discard jars:• Clear phenolic disinfectant such as Stericol or Hycolin. Stericol is available from

AJ Beveridge, 5 Bonnington Road Lane, Edinburgh EH6 5BP, tel. 0131 553 5555.For dealing with spillages:• Clear phenolic disinfectant such as Stericol or Hycolin should be available for

dealing with spillages. A spills kit should be on hand whenever microbiologicalwork is carried out. Advice on dealing with spillages can be found in a suitableCode of Practice.

Advice on lab coats/eye protectionLab coats should be worn for all microbiology practicals.

Plastic aprons do not afford adequate protection as they do not absorb the spilledmaterial but simply redirect it.

Eye protection should be worn in accordance with local guidance from employers.

Gloves of appropriate type for working with stains.



2: POURING PLATES

Materials requiredMaterials required by each student:Lab coatEye protectionDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectant3 bottles of sterile nutrient agar3 sterile plastic Petri dishesBunsen burner and mat

Materials to be shared:Benchkote if necessaryWater baths with water at 55°C (1 or 2, depending on class size).Fine tipped indelible marker pens.Autoclavable bags (or bucket of clear phenolic) for disposal of contaminated plates.

Preparation of materialsAgar – from Oxoid or other standard scientific supplier.

Instructions for the preparation of 100cm3 agar are given below. These can be scaledup to the appropriate volumes.

Preparing agarMaterialsNutrient agar powder Cotton woolSpatula Aluminium foilWeighing boat Bunsen burner, tripod and matWeighing scales 5 universal bottles and lidsDistilled water Autoclave tapeMeasuring cylinder Water bathConical flask Marker penHeat resistant glove


Instructions

1. Wear eye protection and use heat resistant gloves.2. Measure out the required weight of nutrient agar powder to make up 100cm3

‘solution’ (see container for correct weight).3. Add 100cm3 distilled water to a conical flask.

4. Add agar powder to the cold distilled water and swirl around.

5. Make cotton wool stopper.

6. Place stopper in neck of conical flask and cover with aluminium foil (this prevents

the cotton wool catching fire when over a Bunsen flame).

7. Place the conical flask containing the agar in a water bath.

8. Heat over a Bunsen flame, gently swirling occasionally (Use a heat resistant

glove) until it dissolves fully. (N.B. The solution must be heated until the

liquid is transparent; otherwise the agar will not set evenly).

9. Pour the molten agar into universal or McCartney bottles leaving a space of 1–

2cm at the top.

10. Put caps on loosely.

11. Place in basket for sterilisation.

12. Sterilise in a pressure cooker or autoclave under pressure at 15psi (pounds per

square inch).

13. Tighten lids after autoclaving when cool.

The agar can be stored in this form for several months.

When required, lids should be loosened slightly and bottles of agar should be heatedto 100°C until molten then placed in a water bath at 55°C. It is important that thewater level in the water bath lies above that of the agar to prevent solidification.To pour the agar, each student should collect a bottle from the water bath.

Note: To prepare large quantities of agar plates for classes, agar may be prepared asfollows.

Add the appropriate quantity of agar powder to up to 500cm3 water in a bottle andautoclave. Pour plates directly from the bottle when the agar has cooled to theappropriate temperature. Volumes greater than 500cm3 should not be made as theefficacy of sterilisation procedures cannot be guaranteed for such volumes.



A number of different subculturing techniques are described in the laboratory manual.Materials required for each practical are listed below. Since many of them arecommon to several practicals, the ‘preparation of materials’ section covers all thesubculturing techniques.

Preparation of materials

Nutrient agar platesNutrient agar powder is available from Oxoid or other standard scientific suppliers.

Plates can be poured from flasks of sterilised agar cooled to 55°C using asepticprocedures (see Activity 2, ‘Pouring plates’).

Malt agar platesMalt agar is available in powder form from Oxoid and other standard biologicalsuppliers.

Nutrient broths

Nutrient or peptone broth powder is available from Oxoid and other standardscientific suppliers. Instructions as to quantities to be added to deionised or distilledwater will be found on the containers. The broth should be made up and dispensed in10 cm3 volumes to universal bottles, then sterilised by autoclaving.

Bijoux bottles of sterile waterPlace 2 – 3 cm3 of distilled water in Bijoux bottles and sterilise by autoclaving.Tighten lids.

Sterile swabs

Wrap 2 or 3 swabs facing the same direction loosely in foil and seal with autoclavetape. Sterilise by autoclaving.

Plate cultures of bacteria and yeast to be used as inoculaPlate cultures to be used as inocula should have well isolated single colonies. Thiscan be achieved by following the instructions in the student lab manual for Activity3.2 ‘Subculturing – streak plate inoculation’. Plate cultures can be stored for up tofour weeks in a refrigerator or two weeks at room temperature providing they do notshow any signs of contamination.

Plate cultures of Penicillium and Mucor to be used as inoculaPlate cultures of the fungi to be used should be prepared by inoculating the centre ofa sterile malt agar plate with a fragment of fungus from the stock culture. This can beachieved using a sterile wire loop or needle or by using a sterile scalpel as describedin the student lab manual for Activity 3.3. ‘Plate to plate culture of fungal mycelium’.


Fungal mycelium will grow out from the centre of the plate followed by theproduction of spores. Plate cultures of fungi can be stored for up to four weeks in arefrigerator or one week at room temperature providing they do not show any signs ofcontamination. Young cultures should be provided for the students so that they canidentify the mycelium rather than spores to use for their inoculations.

Only pure cultures should be provided to the students as inocula. Contaminatedplates should be discarded.

Broth cultures to be used as inoculaPure broth cultures must be provided as inocula. These can be prepared by followingthe instructions in the student lab manual for Activity 3.1.3, ‘Loop transfer – solid toliquid’. It is very important that a well isolated single colony is used as the inoculumto minimise the risk of contamination. Broth cultures should be stored for no morethan a week and be used only once since it is not possible to identify from theirappearance whether or not they are contaminated.

Disposal of cultures and contaminated materials

Cultures should be sterilised by autoclaving and disposed of as soon as possible afteruse in accordance with a suitable Code of Practice.



Materials requiredMaterials required by each student:Lab coatBunsen burner and matDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectant1 plate culture Micrococcus luteus with isolated single colonies1 plate culture Saccharomyces cerevisiae (yeast) with isolated single coloniesWire loop4 plates of sterile nutrient agar



Materials requiredMaterials required by each student:Lab coatEye protectionBunsen burner and matDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectant1 broth culture Micrococcus luteus1 broth culture Saccharomyces cerevisiae (yeast)Wire loop4 plates of sterile nutrient agar





Materials requiredMaterials required by each student:Lab coatEye protectionBunsen burner and matDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectant1 plate culture Micrococcus luteus with isolated single colonies1 plate culture Saccharomyces cerevisiae (yeast) with isolated single coloniesWire loop4 universals of sterile nutrient broth



Materials requiredMaterials required by each student:

Lab coatEye protectionBunsen burner and matDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectant1 broth culture Micrococcus luteus1 broth culture Saccharomyces cerevisiae (yeast)Wire loop4 universals of sterile nutrient broth2 bottles sterile water





Materials requiredMaterials required by each student:Lab coatBunsen burner and matDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectant1 plate culture Micrococcus luteus with isolated single colonies1 plate culture Saccharomyces cerevisiae (yeast) with isolated single coloniesWire loop4 plates of sterile nutrient agar




Lab coatEye protectionBunsen burner and matDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectant1 broth culture Micrococcus luteus1 broth culture Saccharomyces cerevisiae (yeast)Wire loop4 plates of sterile nutrient agar




Materials requiredMaterials required by each student:Lab coatEye protectionBunsen burner and matDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectant1 plate culture Penicillium roquefortii or Mucor heimalisSmall volume of ethanol in a covered beakerScalpel2 plates of sterile malt agar


Benchkote if necessaryIncubator at 30°CAutoclavable bags for disposal of contaminated platesFine tipped marker pens



Lab coatBunsen burner and matSterile swabsDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectant1 plates of sterile malt agarI plate sterile nutrient agarBijoux bottle of sterile water


Benchkote if necessaryIncubator at 30°CAutoclavable bags for disposal of contaminated platesFine tipped marker pensSellotape


Activity 4: Separating micro-organisms

Materials requiredMaterials required by each student:Lab coatBunsen burner and matDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectant1 mixed broth culture of Phaffia (a red yeast) and Saccharomyces (a creamy whiteyeast)Wire loop2 plates of sterile nutrient agar

Materials to be shared:Benchkote if necessaryAutoclavable bags for disposal of contaminated plates.Fine tipped marker pens


Sterile nutrient agar plates

See ‘Technical Guide’ for ‘Subculturing micro-organisms’.

Mixed broth culture of Phaffia and SaccharomycesIt is best to grow pure broth cultures of each organism and mix them asepticallyimmediately prior to student use rather than inoculate one broth with both organisms.Note that Phaffia grows at room temperature, not 30°C. Saccharomyces will grow atroom temperature also.

It is sometimes difficult to achieve separation from a broth culture using theseorganisms. As a back-up, mixed plate cultures with isolated colonies can be preparedfor students to further subculture. These plates are best prepared by streaking out apure culture of Phaffia and then streaking a pure culture of Saccharomyces along thesame inoculation lines. After incubation at room temperature, these should showisolated colonies of each type which can be used to streak new plates.

If Phaffia is not available, use Micrococcus luteus and E coli as organisms in themixed culture.



5. MICROSCOPIC EXAMINATION OF MICRO-ORGANISMS


Materials requiredMaterials required by each student

MicroscopeBench lampPrepared slides of micro-organisms such as Amoeba and ChlorellaLens tissue

Preparation of materialsBasic care of the microscope is described in the teacher/lecturer guide.

Activity 5.3: Microscopic examination of yeast, pond water and hay infusion(hanging drop)

MaterialsMaterials required by each studentLab coatDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectantLens tissueCavity slide and coverslipVaselineCocktail sticksPasteur pipette (plugged) and bulbPond water and hay infusion


Preparation of materialsA fresh solution of yeast should be made up in nutrient broth.

Pond water can be collected from a non-contaminated pond or stream. Some plantmaterial should be collected if possible as this is a rich source of micro-organisms.

A hay infusion is prepared by placing three or four pieces of clean dry hay or lawncuttings in a Petri dish and covering them with pond or stream water from a non-contaminated source. The lid is then replaced and the dish is kept at room temperaturefor one to two weeks away from direct sunlight.


Activities 5.3.1, 5.3.2 and 5.3.3: Bright field, dark field and phase contrastmicrocopy

If available, microscopes should be set up for dark field and phase contrastmicroscopy according to manufacturers’ instructions.


Lab coatDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectantMicroscopeCavity slide (hanging drop) preparation


Activity 5.4: Calculation of specimen size using a microscope

Materials requiredMaterials required by each student

Microscope set up with eyepiece micrometerStage micrometer slidePrepared slides of micro-organisms to be measured e.g. Paramecium, Euglena, yeast.Lens tissue


To insert the eyepiece micrometer, remove the eyepiece from the body tube, unscrewthe top lens, drop in the micrometer and screw back the top lens. Replace theeyepiece in the body tube.


6: STAINING

Further information regarding the safety of stains can be obtained from SSERC’sHAZARDOUS CHEMICALS MANUAL and CD.

Activity 6.1: Preparation of a smear of bacteria or yeast from a solid culture.

MaterialsMaterials required by each studentLab coatEye protectionDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectantLens tissue3 glass slidesLabelsPlate cultures of yeast and named bacteria with single coloniesLoopForcepsBijoux bottle of sterile waterBunsen burner and mat



Activity 6.2: Observation of bacteria using the simple stain methylene blue

MaterialsMaterials required by each studentLab coatDisposable glovesDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectantFixed smears of Spirillum, Bacillus subtilis and Micrococcus luteus and yeastMethylene blue stainBlotting paper (fibre free)Distilled water bottleStaining rack and dishForcepsMicroscopeImmersion oil




Preparation of materialsThe fixed smears can be prepared according to instructions in Activity 6.1 in thestudent guide. Staining racks can be made from two glass rods joined at each endwith rubber tubing (Fig.1 in teacher/lecturer guide).

Methylene blue stain (for dead organisms)5g methylene blue3g trisodium citrate3.4g sodium chloride500 cm3 distilled water

Mix well then filter.

Activity 6.3: Observation of the filamentous fungi, Mucor and Penicillium usingLactophenol Blue.

Safety noteLactophenol cotton blue contains phenol and is poisonous by absorption. Wear wellfitting nitrile gloves when preparing the stain. Disposable gloves need not be nitrilewhen preparing slides.


Lab coatDisposable glovesDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectantPlate cultures of Mucor and Penicillium2 slides and coverslipsLens tissueLactophenol blueForceps & mounted needleMicroscopeBunsen BurnerMat


Preparation of materialsThe plate cultures should be 5- 7 days old to allow the observation of mycelium.

Lactophenol cotton blue20g phenol20 cm3 lactic acid40 cm3 glycerol0.05g cotton or methyl blue20 cm3 distilled water


Activity 6.4: Negative staining of yoghurt with nigrosin

MaterialsMaterials required by each studentLab coatDisposable glovesDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectantLive yoghurtSmall beakerNigrosin stainLens tissues3 glass slidesWire loopSterile water


Preparation of materialsNigrosin should be made up 5g in 100 cm3 of distilled water. It must be stirred forseveral hours. Correct preparation of nigrosin is critical to successful use.

Activity 6.5: Staining and observation of Rhizobium in root nodules

MaterialsMaterials required by each studentLab coatDisposable glovesDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectant3 glass slidesLens tissuesWire loopForcepsSterile distilled waterRoot nodules on legume rootsCrystal violet stainStaining rack and dish




Preparation of materialsRoot nodules can be obtained from pea seedlings grown in nitrogen rich soil.

Crystal violetDissolve 0.5g in 100 cm3 distilled water

6.6: Vital staining

MaterialsMaterials required by each studentLab coatDisposable glovesDisinfectant and paper towelsDiscard jar with freshly diluted clear phenolic disinfectantBunsen burner and matLens tissuesGlass slide and coverslipWire loopSterile waterPlate culture of yeastNeutral redBlotting paperMicroscope



Neutral redDissolve 0.1g neutral red in 1 litre isotonic saline

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Biotechnology working with microoranisms

Engineering