Microbiology Laboratory Manual

Practical 03
Practical: Identification of bacteria using Simple Staining Technique
Objectives: 1. Prepare a smear from a bacterial specimen
(a) Sample taken from a solid agar medium
(b) Sample taken from a broth culture
2. Prepare a simple stain of the smear
(a) Positive staining
(b) Negative staining
3. Use the microscope to identify features (shape, arrangement and size) of the
bacterium.
Introduction:
In order to stain the bacterial specimen for microscopy one must first prepare the
smear onthe slide. This basically involves 3 steps----transferring a liquid suspension
of the bacteriumon the slide, drying the smear and then heating slightly to firmly
attach the smear to the slide. Once this is done, the staining procedure begins.
CAUTION: Do NOT make your smear suspensions too thick: The dye will not
penetrate well and there will be far too many bacterial cells to see individual shapes
and arrangements.

Methodology:
Materials Needed:
NB culture of Staphylococcus epidermidis
NA plate of E. coli
Dye reagents/ Methylene blue
Clean microscope slides/ Cover Slips
Wax pencil
Blotting papers

The Procedure:
1. (a) If the culture is taken from solid (agar) medium

i. Clean the slide using detergent. Gently dry the slide with a clean cloth.
(alternatively, it is possible to use pre-cleaned slides)
ii. Place a very small drop of distilled water on the surface of the slide

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iii. Aseptically remove a small amount of the culture from the agar surface and
just touch it several times to the drop of water until it just turns cloudy
iv. Burn the remaining bacteria off of the loop. (If too much culture is added to
the water, you will not see clearly stained individual bacteria)
v. Using the loop, spread the suspension over the entire slide to form a thin film
vi. Allow this thin suspension to completely air dry
vii. Pass the slide (film-side up) through the flame of the Bunsen burner 3 or 4
times to heat-fix. Caution: Too much heat might distort the organism. The
slide should feel very warm but not too hot to hold

1. (b) If the organism is taken from a broth culture:

i. Clean the slide using suitable cleaning material. Gently dry the slide with a
clean cloth. (alternatively, it is possible to use pre-cleaned slides)
ii. Aseptically place 2 or 3 loops of the culture on a clean slide. Do not use water
iii. Using the loop, spread the suspension over the entire slide to form a thin film
iv. Allow this thin suspension to completely air dry
v. Pass the slide (film-side up) through the flame of the Bunsen burner 3 or 4
times to heat-fix

2. (a) Simple (Positive) Staining with Methylene Blue

i. Prepare a heat fixed smear of the culture you wish to examine

ii. Cover the smear with methylene blue
iii. Allow the dye to remain on the smear for approximately 1 minute. (Note
staining time is not critical. Somewhere between 30 seconds and 2 minutes
should give you an acceptable stain. The longer you leave the dye on, in
general, the darker the stain)
iv. Wash the excess stain off the slide (Pick up the slide by one end and hold it at
an angle over the staining tray. Using the distilled water wash bottle, gently
wash off the excess methylene blue from the slide by directing a gentle stream
of water over the surface of the slide. Wash off any stain that got on the
bottom of the slide as well

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v. Blot off excess stain using blotting paper. DO NOT rub the slide, rather place
the slide between two sheets of blotting papers and press down gently. Paper
will absorb excess dye
vi. Examine the slide under the light microscope
vii. Record the shape, arrangement, and approximate size of the organisms. You
should have data on two cultures which you have stained

2 (b) Negative (Indirect) Stains

i. Clean the slide using suitable cleaning material. Gently dry the slide with a lint
free cloth. (alternatively, it is possible to purchase pre-cleaned slides)
ii. Place a small drop of the negative stain (India ink) near the end of the slide
iii. Transfer one loopfull of the bacterial sample to the india ink and mix the two
together
iv. Hold a clean slide at about a 20o angle to the first slide. Touch the edge of the
clean slide to the bacteria/stain mixture so that the mixture spreads across the
edge
v. Spread the suspension across the surface of the slide by drawing the clean
slide away from the mixture. Essentially, you will be using the clean slide to
push the mixture across the surface of the slide. When you have finished
spreading the slide, place the "clean" slide in a jar of disinfectant
vi. Air dry the slide. DO NOT HEAT FIX
vii. Examine under the microscope
viii. Record the shape, arrangement, and approximate size of the organisms. You
should have data on two specimens which you have stained

QUESTIONS:

1. Record your results of staining

2. Since everything on the slide will be the same color in simple staining, what you
possibly tell about the organisms?
3. Are all bacteria the same size?
4. What names are given to the shapes seen in the bacteria used in lab?
5. Why heat-fix the smear?
6. Why are basic dyes more useful than acidic dyes when staining bacteria?
7. What is the disadvantage of having a really thick smear when staining?

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