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CBB 20003 Principles of Microbiology

Experiment 2
Media Preparation and Grams Staining
Good microbiological laboratory practice:
Any exposed cuts and abrasions should be protected with waterproof dressings before
the practical work starts.
Wash your hands before and after practical work.
The laboratory door and windows should be closed when work is in progress. This
will reduce air movements and consequently the risk of accidental contamination of
your cultures.
Laboratory coat must be worn.
High standards of cleanliness must be maintained. Non-porous work surfaces should
be used and they should be swabbed with an appropriate laboratory disinfectant (70%
ethanol) before and after each practical session.
Cultures should not be removed from the laboratory.
PART 1: MEDIA PREPARATION
Pre-lab Reading
Growth media
The culture medium must supply all the essential nutrients needed by the
microorganisms. The medium used depends on the microorganism that one is trying to isolate
or grow. The pH of the medium should be adjusted to that preferred by the organism under
study. If these requirements are met, and the incubation conditions such as temperature, and
the degree of aeration are favorable, good growth should result. There are both liquid and
solid growth media.
Solid media contain a gelling base plus the nutrients needed for growth. Agar is the
most commonly used gelling agent. It dissolves in water at 100 C and sets at around 48 C.
It should be kept at 50 C oven until ready for use, and then used quickly once removed. For
this reason, molten growth media should not be pipetted. Microorganisms deposited on solid
media will multiply under appropriate incubation conditions. One microorganism will
eventually produce a colony on the agar plate. An agar slant is simply a tube placed at an
angle during cooling to give a large slanted surface for inoculation. They are commonly used
for the preservation of stock cultures.
A liquid medium (broth) is used for boosting growth. The convection currents in a
liquid prevent overcrowding and the buildup of waste products by dispersing the
microorganisms. If a mixed culture is grown in liquid media, it is difficult to isolate an
individual species, so it is impossible to obtain a pure culture from such sample. It is
necessary to subculture onto a solid medium in order to isolate the pure culture.

CBB 20003 Principles of Microbiology

Containers
These are needed to store sterile media and to grow cultures in. The most frequently used are:

Petri dishes are used to cultivate microbes on solid nutrient media. Sterile plastic
dishes are used once, then discarded along with their contents after use. Glass Petri
dishes can be reuse after autoclaving. A Petri dish hold 15 to 20 ml of medium.
Universal bottles are glass screw capped jars of 20-30 ml capacity. They are used for
holding stock cultures, deeps, slopes and molten agar for pouring plates.
Conical flasks are used for growing microbes in liquid culture media and also for
holding large volumes of sterile media.

Methodology
(A) Preparation of nutrient agar (250 ml for 10 plates and 5 agar slants)
1. Weigh the nutrient agar powder required according to the manufacturers
instructions.
2. Mix the nutrient agar powder in 200 ml of distilled water. Put in the stir bar and
turn on the stir plate. Apply heat to the mixture. Upon boiling, the agar dissolves,
it will turn clear, deeper tan. Remove the agar from heat.
3. Transfer 10 ml of agar into 5 universal bottles respectively (for agar slants).
4. Pour the remaining agar into Schott bottle.
5. Autoclave the medium at 121 C for 15 minutes.
6. For agar slant preparation, while the medium is still hot after autoclaving, tilt the
bottles with agar on a solid surface so that the medium in the bottles are slanted.
Allow the medium o harden in this position. When the medium is cool, tighten the
caps. Keep the agar slants at 4 C for Practical 3.
7. Follow the protocol in Section (B) for preparing nutrient agar plates.
(B) Pouring a plate
1. Wipe the laminar cabinet with 70% ethanol, and have a Bunsen burner on with a
roaring flame. Get ready the sterile Petri dishes and a Schott bottle of molten
nutrient agar.
2. Put the sterile Petri dish near your right hand with the lid uppermost. Do not open
the Petri dish until ready to pour the agar. Loosen but do not remove the cap of
Schott bottle.
3. Hold the Schott bottle in your right hand. Remove the cap.
4. Flame the neck of the bottle by passing it briefly through the flame. Using the left
hand, lift the lid of the Petri dish slightly and pour molten agar onto the base.
5. Replace the lid of Petri dish. Gently rotate the base of the Petri dish so that the
agar forms a continuous later over the base. Leave the plate in the cabinet to set.
6. Keep the agar plates at 4 C for Practical 3.

CBB 20003 Principles of Microbiology

PART 2: GRAMS STAINING


Pre-lab Reading
The Gram stain is the most widely used staining procedure in bacteriology. It is called
a differential stain since it differentiates between Gram-positive and Gram-negative bacteria.
Bacteria which stain purple with the gram staining procedure are termed gram-positive; those
which stain pink are said to be gram-negative. The terms positive and negative have nothing
to do with electrical charge, but simply designate two distinct morphological groups of
bacteria.
Methodology
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Place a drop of the sterile distilled water on a clean glass slide.


Transfer a small loop of the colony from the plate into the drop and mixed them
gently and spread over the slide.
Allow the smear to dry by gentle heating.
Flood the smear with crystal violet solution for 30 seconds, followed by washing the
stain with iodine for 30 seconds.
Drain off the excess iodine and wash with running 70% ethanol for 5 seconds. Rinse
the slide with running tap water and counterstain with safranin for 30 seconds.
Wash the slide again with running tap water and blot to dry in the air.
Examine the slide under the microscope in oil immersion using the highest
magnification. Observe the stained cells; gram positive is shown by purple cells,
while red cells indicate gram negative.

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