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This article will guide you about how to study bacteria.
Bacteria are minute organisms of enormous number with a high rate of multiplication. The smallest known bacterium is a little over 1 μm in size. Extreme precaution should be taken while handling bacteria—particularly pathogens (disease causing). Maintenance of an aseptic condition is a must in bacteria studies.
I. Hanging drop method:
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1. Take a clean grease-free glass slide. Ring it at the middle with Vaseline (use turn table).
2. Put a drop of material to be examined on a clean cover slip.
3. Place the cover slip inverted on the slide fitting the ring.
4. Examine the slide under low/high power magnification of a compound microscope.
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II. Wet preparation of stool: motility study:
1. Take a glass slide and make a thin suspension of a drop of normal saline solution on the slide. Take the material to be examined with a sterile (heated red in a flame and cooled) platinum loop and put it on the slide.
2. Put a cover slip over the suspension and drain the excess fluid with filter paper. Examine under microscope. A Vaseline-ringed cover slip may be used to prevent drying.
III. Preparation of film for staining:
Take a grease-free clean glass slide. Flame the platinum loop and allow it to cool. Take a loopful of broth culture/bacterial suspension/ the substance to be examined (urine, sputum, or any other material); put it at the middle, and make a thin smear. Dry in air. Fix it by rapidly passing 2 or 3 times over the flame, taking care that it is- not overheated or charred. Mark the slide to indicate the surface on which the smear is made.
IV. Staining of film : Gram’s method (Jenson’s modification) (Fig. 27.1)
1. Place the slide with the fixed smear on a staining rack.
2. Cover the smear with few drops of 1 -0% aqueous methyl violet for one minute.
3. Rinse the stain with water for 2 seconds.
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4. Wash off with Gram’s iodine for mordanting, wait for a minute.
5. Differentiate with alcohol for 5 seconds till the smear is faintly violet.
6. Rinse with water for 2 seconds.
7. Look for bacterial patches. If required, treat again with ethanol for 5 seconds and rinse well with water and drain off water.
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8. Counter stain with 1% aqueous safranin stain for 40 seconds.
9. Rinse with water for 2 seconds.
10. Blot off water and allow to dry in air.
11. Examine under microscope with oil immersion objective.
Observation:
The bacteria can be identified or classified by their reaction to the stain. Iodine fixes methyl violet to Gram positive cell and the violet colour is not washed out by alcohol. Gram negative cells appear pink because alcohol washes out the violet colour before the smear is stained by safranin.
Gram +ve bacteria:
Bacillus, Staphylococcus, Streptococcus, Micrococcus, Lactobacillus, Corynebacterium, Clostridium.
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Gram -ve bacteria:
Cholerae, Salmonella, Shigella, Pseudomonas, Klebsiella, Escherichia, Enterobacter, Brucella, Haemophilus, Neisseria, Proteus.
