Student Lab Guide - 2 Hour Labs - Part 2

7/31/2019 Student Lab Guide - 2 Hour Labs - Part 2

1/23

23

Lab 8

Exercise 18 Culture Media Preparation Part 2

Background:Culture Media nutrients used to grow cells or microbes

A. Agar nutrients in gel-like formPlates, slants, and deeps

B. Broth nutrients in liquid form

Procedure:Weigh out then mix media with water in a flask that is at least twice the capacity of the volume of

the water that is used.

If media is an agar, heat to boiling for 1 minute, stirring constantly, to dissolve. Set hot plate to "10".

Broths do not require heating.

Boiling media will foam up, so be ready with a hot pad to quickly remove media from hot plate.

Dispense required volume into tubes using a pipet. If preparing plates, just cap flask with foil.

Put caps on tubes.

For tubes, write 4 labels with class, type of media, and date, and then put on top of caps. Use a pencil.

For flasks used for plates, write 2 labels with class, type of media, and date, and then put one on foil

and one on flask.

For "bottle media", use the screw-cap square bottles. Just place the caps on the bottles. Do not

screw the caps on.

Put the media in the spot designated by the instructor.

Instructor will autoclave media.

Rinse out pipettes with water using pipette pumps.

Group Media MediaVol.Water

TotalNo. Vol. / t

(g) (mL)Tubesor

(mL /tube)

Plates

to Make

1

Trypticase Soy Broth 2.3 75 15

Fluid thioglycollate Agar Deep 1.4 48 8

Trypticase Soy Agar Plates 8.0 200 8

2Trypticase Soy Agar bottles 21.0 525 7

EMB Agar Plates 14.0 375 15

3MSA Agar Plates 41.6 375 15

MacConkey Agar Plates 10.0 200 8

4

Phenol Red Glucose Broths 1.5 75 15

Phenol Red Lactose Broths 0.8 40 8

MR-VP broths 1.3 75 15

5

Simmons Citrate Agar Slants 1.5 60 15

Mueller-Hinton II Agar Plates 7.6 200 8

90 mL sterile water bottles 0.0 630 7

6

99 mL sterile water bottles 0.0 693 7

Soft Agar Deeps (use "Agar, granulated") 0.3 48 8

Motility Media Agar Deeps 1.1 48 8


2/23

24Lab Report: none

Know for Exam:

Know how to weigh, pipette, mix, and dispense media. Do not have to memorize media recipes.

Know how to pour plates.

Know the different types of culture media preparations.

Dilutions

Preparation:

Background:

Procedure:Take notes on Dilutions

Know for exam: Be able to solve dilution problems Be able to calculate concentrations of bacteria in stock samples

Exercise 18 Culture Media Preparation Part 2

Procedure:Each group label plates as indicated below on bottom with a wax pencil & pour.Rinse out empty flasks, remove labels, and put away.

Group Number of plates Label1 8 TSA (trypticase soy agar)2 15 EMB (eosin methylene blue agar)3 8 MSA (mannitol salt agar)4 7 MSA (mannitol salt agar)5 8 MH (Mueller-Hinton II agar)6 8 Mac (Maconkey agar)


3/23

25

Lab 9

Exercise ?? Selective and Differential Media Inoculations (not in labbook)

Background:Selective Media

Selects (promotes growth of) some bacteria, inhibits othersDifferential Media

Differentiates different microbes based on appearance

Procedure:A. Each group label the following plates on bottomwith wax pencil

EMBMSAMacConkey

Also include paper label with regularinformation (section no., etc.)

B. Aseptically inoculate EMB, MSA, and Mac plates with1. E. coli2. Proteus mirabilis3. Staphylococcus epidermidis4. Bacillus subtilis

E. Incubate all plates 48 hours at 37C

1 24 3


4/23

Summary of Selective and Differential Media

Media Selective Properties Differential ProPromotes Inhibits Appearance

MacConkeyAgar

Gram negative Gram positive Pink colonies

White or gray colonies

Mannitol SaltAgar

Halotolerant Non-halotolerant Yellow medium

Original pink medium

EMB Agar Gram negative Gram positive Metallic green colonies

Dark purple/black colonies

Colonies that are light pink orcolor of media

*Note that there would have to be visible growth in order to determine whether or not bacteria ferm


5/23

27

Exercise 44 Bacterial Counts of Foods Inoculations

Preparation: read page 317 318 and review dilutions notes

Procedure:Follow labbook procedure except:

# 1. Weigh 10 g or 10 mL of food (instead of 20) in sterile Petri plates. # 2. Instead of putting food in blender, add it to 90 mL sterile water

blank and shake 25 times (this is still 1:10 dilution)

Using sample in 90 mL water blank (blender), make streak plates onEMB and MSA.

Clean water blanks, agar bottles and caps with a brush, soap, andwater, and then dry and put away.

Food assignments:Group Food1 Hamburger2 Sushi3 Potted meat4 Bagged salad5 Unwashed Fresh vegetable (chopped broccoli florets)6 Washed Fresh vegetable (chopped broccoli florets)

Exercise 21 Bacteriophage (preparation)

Procedure:Each group inoculate one Trypticase Soy Broth with E. coliB


6/23

28

Lab 10

Exercise ?? Selective and Differential Media Evaluations

Procedure:Evaluate media using Summary of Selective and Differential Media.Enter results on Selective and Differential Media Results table.

Lab Report: none

Know for exam: Define selective and differential media. Know information on Summary of Selective and Differential Media table. Be able to interpret results on the media shown below:

MacConkey Agar Mannitol Salt Agar EMB Agar

Photograph by Jeni SharpePhotograph by Jeni SharpePhotograph by Spence Dowlen


7/23

Selective and Differential Media results

Medium Microbe Amount (+++, ++, +, -)and color of Growth

Color Changein Medium

Con

MacConkey 1. E. coli ------------------

2. Proteus mirabilis ------------------

3. Staphylococcus epidermidis ------------------

4. Bacillus subtilis ------------------

Mannitol SaltAgar

1. E. coli

2. Proteus mirabilis

3. Staphylococcus epidermidis

4. Bacillus subtilis

EMB Agar 1. E. coli ------------------

2. Proteus mirabilis ------------------

3. Staphylococcus epidermidis ------------------

4. Bacillus subtilis ------------------


8/23

30

Exercise 44 Bacterial Counts of Foods Evaluation

Preparation: read page 317 318 and review dilution notes

Background:

Example of results:

Choose plate that has between 30 and 300 coloniesCount even small coloniesCalculate organisms per mL

Organisms/ml = # colonies X inverse of dilution

Selective and Differential media resultsMSA Halotolerant, mannitol fermenter

often Staphylococcus aureus(food poisoning and Staph.infections)

EMB shiny, green coloniesE. coli, often from fecal contamination

Procedure: Calculate the concentration of bacteria in your food sample. Evaluate the results of the MSA and EMB plates that were inoculated with the

food sample. Enter results on Bacterial Counts of Food Results table.

Lab Report: do all

Know for exam:

Be able to determine the bacterial concentration in foods by diluting samplesand applying to plates.

Understand the dilution scheme of Figure 46.1 in the labbook.

Be able to interpret results of food samples applied to MSA and EMB agars.

Be able to answer questions about the typical, ideal results for each foodtested in the lab (will not have to give exact numbers) and why we wouldexpect high or low bacterial counts in each of these foods.

1:100 1:1,000 1:10,000

500Colonies

50Colonies

5Colonies


9/23

Bacterial Counts of Food Results

Type of Food Plate Count Dilution Bacterial Concentration(CFU/mL)

MSA Results*

*MSA Halotolerant, mannitol fermenters are often Staphylococcus aureus.**EMB Shiny, green colonies are usually E. coliwhich indicate fecal contamination.


10/23

32

Exercise 21 Determination of a Bacteriophage Titer Inoculations

Preparation: read pages 165 167

Background:

Procedure:Use Figure 23.2 to see an overview of procedure, but note that we will notmake any dilutions and will make only one plate.

1. Label 1 TSA plates with standard 4 items of information.2. Remove 1 liquid soft agar tube from the hot water bath and transfer 1

mL of the phage culture to the tube using a sterile transfer pipette.3. Using the same transfer pipette, transfer 2 3 drops of the E. coliB

broth culture to the soft agar tube and mix by rolling tube in hands.

4. Pour the contents of the soft agar tube onto the TSA plate. Gently rockthe plate so that the soft agar completely covers the surface.

5. Allow the plate to sit for a few minutes so the soft agar can solidify.6. Since the initial bond between the two agar layers is weak, place the

plate right side up in the 37 C incubator. The instructor will turn themupside down in 1 hour.

Photograph by Jeni Sharpe


11/23

33

Lab 11

Exercise 21 Determination of a Bacteriophage Titer Evaluations


Background:

Plaques clear areas of dead bacteria caused by bacteriophageDRAW

Procedure:Examine plates looking for plaques.

Lab Report: B(1-3, 5, 6)

Know for exam: Understand the procedure for preparing this plate of bacteriophage.

Define plaques. Understand lab report material.



12/23

34

Exercise 34 Morphological Study of Unknown Bacterium Inoculations


Background:Study of Unknown Bacterium General Plan

Receive unknown bacteria (Group 1 gets Unknown # 1, etc.)Run tests on it over next several labsCompile results and identify

Procedure:A. Gram stain unknown and observe

Record on Test Results on Unknown table:Gram reactionCell shapeCell arrangementPresence of any endospores

B. Motility

Inoculate motility media by stabbing with inoculating needle about 2/3of the depth of the media.Incubate 1 day at room temperature.

Know for exam:

Know how to perform a Gram Stain on unknown and interpret the followingresults.

o Gram reactiono Cell shapeo Cell arrangemento Presence of endospores

Exercise 35 Cultural Characteristics Inoculations

Preparation: read page 245

Procedure:

Inoculate a tube of fluid thioglycollate media (FTM) with a loopful of theunknown.

Mix organisms by rolling the tube between your palms.

Include type of media (FTM) on label.

Incubate at room temperature.


13/23

35

Exercise 36 Oxidation and Fermentation Tests Inoculations


Background:Oxidation and Fermentation Tests

Testing for various fermentation products and enzyme production

Test that will be performed:A. Glucose fermentationB. Lactose fermentationC. Mixed acid fermentation (MR test)D. Citrate testE. Catalase test

Procedure:

Inoculate the following media with the unknown:o Phenol Red Glucoseo Phenol Red Lactoseo MR-VPo Citrate

Inoculate the following media with known bacteria to use as positive testcontrols:

o E. coli Phenol Red Glucose MR-VP

o Enterobacter aerogenes Citrate

Include name of media on each tube.

Each group will need:o 2 Phenol Red Glucoseo 1 Phenol Red Lactoseo 2 MR-VPo 2 Citrate

Some types of media look identical. Label or mark media before take back totable.

Put tubes in incubator, consolidating tubes into as few racks as possible.


14/23

36

Exercise 31 Antibiotic Sensitivity Testing (preparation)

Procedure:Each group inoculate one Trypticase Soy Broth as follows:

Group Bacteria1 Bacillus subtilis2 E. coli3 Proteus mirabilis4 Enterobacter aerogenes5 Bacillus subtilis6 E. coli

Incubate at 37 C


15/23

37

Test Results on Unknown

Unknown # ________________ Bacterial ID _______________________________

Observations Conclusions

Gram Reaction

Shape

Arrangement

Presence of Endospores

Motility Test

Oxygen Requirements

Glucose Fermentation

Lactose Fermentation

Mixed Acid Fermentation(MR Test)Citrate Test

Catalase Test


16/23

38

Lab 12

Exercise 34 Morphological Study of Unknown Bacterium Evaluations

Preparation: read page 242 Motility

Procedure:Evaluate motility media tube for motilityand record on Test Results onUnknown table.

Know for exam:

Know how to determine the motility ofan unknown using motility media.

Exercise 35 Cultural Characteristics Evaluations

Preparation: read page 247

Background:Using fluid thioglycollate media (FTM) to determine oxygen requirements:

Thioglycollate binds available oxygenFTM has an indicator that turns green if oxygen presentThere is more oxygen at top of media, none at the bottom

DRAW

Obligate Facultative ObligateAerobe Microaerophile Anaerobe Anaerobe

Procedure:

Examine thioglycollate tube, determine and record oxygen requirements onTest Results on Unknown table.

BE GENTLE WITH TUBES. DO NOT SHAKE OR MIX

Know for exam:

Know how fluid thioglycollate media works and be able to interpret results Only drawings of growth distributions will be used on the exam.



17/23

39

Phenol RedLactose Broth MR-VP Media

SimmonsCitrate Agar

Exercise 36 Oxidation and Fermentation tests Evaluations

Preparation: read pages 249 258, and Diagnostic Media Summary

Procedure:

Do Second Period Test Evaluations for:o Carbohydrates in Durham Tubeso Mixed Acid Fermentationo Citrate Testo Catalase test (use unknown slant and Proteus mirabilisstock cultures.)

Use Diagnostic Media Summary Table to help determine results.

Record results for unknown on Test Results on Unknown table as positiveor negative.

Know for exam:

Know information on Diagnostic Media Summaryand be able to determine the following when giveninoculated media:

o Testo Mediao Substrateo Enzymeo Producto Any added reagentso Whether test is positive or negative

On exam, will be told either what media is beingused or what test is being run.

Phenol RedGlucose Broth

Photographs by Karen Kendall-Fite

Photographs by Karen Kendall-Fite Photographs by Karen Kendall-Fite

Photograph by Karen Kendall-Fite


18/23

Diagnostic Media Summary

Test Media Reactions Occurring in Positive Tests*(Substrate enzyme Product)

IndPo

GlucoseFermentation

Phenol RedGlucose broth

Glucose glucose fermentation enzymes acids Ye

LactoseFermentation

Phenol RedLactose broth

Lactose lactose fermentation enzymes acids Ye

Mixed AcidFermentation

MR-VP Glucose mixed acid fermentation enzymes strong acids+ Methyl Red

Re

Citrate Test Simmons CitrateAgar slant

Citrate citrase alkaline products Blu

Catalase Test Nutrient agarslant

H2O2catalase

O2 + H2O Bu

*Bold compounds are reagents added by experimenter.

Be able to recognize positive and negative reactions and to name the substrate, enzymes, productreaction.


19/23

41

Exercise 39 Use of Bergeys Manual (Identification of Unknown Bacterium)


Background:Bergeys Manual

Best classification schemeDivide bacteria up into 11 groupsSeveral genera, many species in each group

Procedure:

Use Figures 41.1 and 41.2 to identify group of unknown

Use text descriptions to identify genus

Check answer with instructor

Know for exam:

Given tables and information on pp. 275 279 and lab results in a TestResults on Unknown table, be able to determine genus of unknown bacteria.

Exercise 31 Antibiotic Sensitivity Testing Inoculations


Background:Antibiotic Sensitivity Testing: The Kirby-Bauer Method

Measures the sensitivity of bacteria to an antibiotic by measuring howwell growth in inhibited (zone of inhibition)DRAW

Procedure:

Follow labbook procedure for First Period plate preparation except use brothinoculated in last lab to make lawns.

Applicator will lock up if one of the cartridges is empty. Do not forcemechanism. (Replace empty cartridge and then continue.)

Gently press down discs with sterile loop (WCC and LCC)


20/23

42

Lab 13

Exercise 31 Antimicrobic Sensitivity Testing Evaluation


Procedure: Follow labbook Second Period Interpretation

The letter codes for the antibiotic disks are as follows:Tetracycline (TE) Sulfisoxazole (SXT or G) Streptomycin (S)Gentamicin (GM) Ampicillin (AM) Ciprofloxacin (C)

Measure diameter of the zones of inhibition (ZOI) from bottom of plate.

If no inhibition, just record diameter of disc (7 mm)DRAW

Use Table 33.1 to determine if bacteria are resistant, intermediate, orsusceptible to the antimicrobial.

Enter results on table on p. 221.

If bacteria is not covered in Table 33.1, leave blank

Know for exam:

Be able to use the Kirby-Bauer method to determine whether bacteria aresensitive, intermediate, or resistant to an antibiotic.

On the exam, you will be given a plate, ruler, Table 33.1, and the letter codefor the antibiotics used.

Photograph by Karen Kendall-FitePhotograph by Karen Kendall-Fite


21/23

43

Lab Cleanup by Students

Procedure:

Clean bench top Clean bench top gutter

Clean out sinks

Clean microscopes (each group clean two) Wash out staining trays

Wrap cords around Bacticinerators

Clean inside and outside of incubator Fill water bottles

Fill disinfectant bottles

At Columbia and Lawrenceburg for each bino Remove trasho Wipe out with disinfectanto Make sure each bin has the following:

1 inoculating needle 2 loops 2 clothespins 1 lens paper booklet 2 half-rack 1 wax pencil 10 sheet of labels 1 ruler

o Put extra drawer items on front desk

At Franklin for each groups drawers

o Remove trash including from shelf above chairo Wipe out with disinfectanto Make sure each station has the following:

Top Drawer 1 inoculating needle

1 loop

1 clothespin 1 lens paper booklet

1 wax pencil

1 immersion oil 1 stirring rod

1 flask clamp 1 test tube clamp

1 weigh boat

6 sheet labels.

1 ruler


22/23

44

Second Drawer 1 pair Hot pad gloves

Cabinet

Burner Rack

Bacticinerator (one each table)

Burner stand. 100 mL graduated cylinder

Open Lab

Procedure:

Review examples of inoculated media. Know all information, procedures, handouts, and lab reports given in the Lab.


23/23

45

Lab 14

Lab Exam # 2

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Student Lab Guide - 2 Hour Labs - Part 2

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