8/8/2019 71 Microscopy

1/40

1- Wear a white coat.

2- Eating and smoking are prohibited.3- Never put anything in your mouth(fingers, papers), use a teat during pipetting.4- Wash your hands after completing yourwork with soap and water and a disinfectant.

8/8/2019 71 Microscopy

2/40

` 1- Report at once if any accident occurs in the laboratory.

` 2- Wash cuts or pricks at once with soap and water and

add an antiseptic e.g. 70% alcohol or 0.5% chlorhexidine.` 3- If the eye is splashed with infective material, rinse it at

once with tap water.

` 4- If the mouth is contaminated, spit and rinse with water.

` 5- If clothing is contaminated moisten the area withstrong phenol and rinse with water.

8/8/2019 71 Microscopy

3/40

` 1- Culture tubes are kept in a rack or a tin.

` 2- Old cultures and contaminated glassware must be

placed in discard tins to be autoclaved.` 3- Contaminated pipettes are put in a jar with 5%

lysol.

` 4- 20% lysol is used for disinfecting drops of culture

that may fall on the bench

8/8/2019 71 Microscopy

4/40

` (A) MICROSCOPY

`The purpose of the microscope is toproduce an enlarged well definedimage of objects too small to be

observed with the naked eye.

8/8/2019 71 Microscopy

5/40

` Amicroscope is a magnifying instrument. The

magnified image of the object (specimen) is

first produced by a lens close to the object

called the objective.

` This collects light from the specimen and

forms a primary image.A second lens near the

eye called the eye piece enlarges the primaryimage, converting it into one that can enter the

pupil of the eye.

8/8/2019 71 Microscopy

6/40

` 1- Heavy foot. 2- Upright limb.` 3- Stage: with a central hole over which the slide with specimen

is held with clips.` 4- Optical system consists of:` An external tube that bears a revolving nose piece where

several objectives are fitted.` An inner tube that carries the eye piece.` 5- Knobs of coarse and fine focusing adjustments.` 6- Substage consists of:` Condenser: to focus the beam of light on the specimen, can be

moved upwards or downwards.` Iris diaphragm: with a lever to open and close the iris as

required.` 7- Hinged mirror for daylight use or built in electric lamp.

8/8/2019 71 Microscopy

7/40

` Total magnification by low power 100

` Total magnification by high power 400

` Total magnification by oil immersion 1000

8/8/2019 71 Microscopy

8/40

8/8/2019 71 Microscopy

9/40

` In most bacteriological work the oil immersion lens isused.A good source of light is needed. The condenseris raised to its upper most position and the irisdiaphragm is fully opened.A drop of cedar wood oil is

placed on the stained smear.`

` The nose piece is rotated until the oil immersion lens isin position. The coarse focusing adjustment is moved

slowly until the oil on the slide makes contact with theobjective and the fine adjustment is used until the filmcomes into view.

8/8/2019 71 Microscopy

10/40

` Dark ground microscope has proved useful for

observing organisms such as spirochaetes,

which are so thin (less than 0.2 in diameter)

and therefore can not be observed with the

ordinary light microscope.

8/8/2019 71 Microscopy

11/40

` 1-UV light of shorter wave length 0.2um.

` 2- Lenses made of quartz.

` 3-Fluorescent dye (auramine,acridine orange,

fluorescein) which absorbs UV invisible rays

and emits visible rays.

8/8/2019 71 Microscopy

12/40

` When it is required to visualize viruses or

structures with a diameter less than 0.2 um

(which is the limit of resolution of the light

microscope), it is necessary to use the electron

microscope.

` Its power of resolution is 0.001 um or 1nm.

8/8/2019 71 Microscopy

13/40

` It is a metal chamber with a thermostat to maintain the

temperature inside at the required degree for incubating

bacterial cultures.

` Incubation is a process for maintaining a bacterialculture at a particular temperature for a certain length

of time in order to measure bacterial growth.

` Almost all bacteria which are pathogenic to man have

an optimum temperature for growth at 370

C (i.e. bodytemperature).

8/8/2019 71 Microscopy

14/40

` Flat, circular and completely closed loop mounted on a

handle. Used as an inoculating instrument by taking up

a considerable amount of solid culture or a large drop

of liquid.` It can be made of platinum, stainless steel or nichrome.

It is sterilized by holding it almost vertically in a

bunsen flame so that the whole length becomes red hot

at the same time.

8/8/2019 71 Microscopy

15/40

8/8/2019 71 Microscopy

16/40

Typical shapes of bacteria

Most bacteria retain a particular shape; a few

are pleiomorphic

8/8/2019 71 Microscopy

17/40

` CoccusCoccus

ChainChain == StreptoccusStreptoccus

ClusterCluster == StaphylococcusStaphylococcus

` BacillusBacillus

ChainChain == StreptobacillusStreptobacillus

` CoccobacillusCoccobacillus

` VibrioVibrio == curvedcurved` SpirillumSpirillum

` SpirocheteSpirochete

8/8/2019 71 Microscopy

18/40

8/8/2019 71 Microscopy

19/40

` Average bacteria 0.2 um in diam. RBC is 7.5 um in diam.

` SurfaceArea ~12 um^2

` Volume is ~4 um` SurfaceArea to Volume is 3:1` Typical Eukaryote Cell SA/Vol is 0.3:1` Food enters through SA, quickly reaches all parts of

bacteria

` Eukaroytes need structures & organelles

8/8/2019 71 Microscopy

20/40

Characteristic grouping (or not grouping)

8/8/2019 71 Microscopy

21/40

Gram +ve cell wall Gram ve cell wall

1- The PG layer is the

major constituent of the cell

wall (50%-90%)

1- The peptidoglycan is thinner only 5-

10% of cell wall.

2- The Gram +ve cell wall

contains also teichoic acid (which is

responsible for the surface antigen

ofG +ve bacteria.

2- External to the PG layer is the

lipoprotein and outer membrane.

8/8/2019 71 Microscopy

22/40

1-Crystal violetp 1`wash with H2O.

2-Grams iodinep

1`wash

3-Decolorize with 95% ethyl alcoholp 1-2`.wash

4-Counter stain with safraninp 1`.wash then dry

8/8/2019 71 Microscopy

23/40

8/8/2019 71 Microscopy

24/40

8/8/2019 71 Microscopy

25/40

8/8/2019 71 Microscopy

26/40

8/8/2019 71 Microscopy

27/40

8/8/2019 71 Microscopy

28/40

8/8/2019 71 Microscopy

29/40

8/8/2019 71 Microscopy

30/40

8/8/2019 71 Microscopy

31/40

8/8/2019 71 Microscopy

32/40

8/8/2019 71 Microscopy

33/40

8/8/2019 71 Microscopy

34/40

8/8/2019 71 Microscopy

35/40

8/8/2019 71 Microscopy

36/40

8/8/2019 71 Microscopy

37/40

` India ink or Negrosin Fuchsin.

` Acidic dye.

` Demonstrate the background.

` Capsular staining.

8/8/2019 71 Microscopy

38/40

8/8/2019 71 Microscopy

39/40

Methylene blue.

Basic dye ( colored cation + ).

Demonstrate shape and arrangement.

8/8/2019 71 Microscopy

40/40