Review Microbiology Lab Test 2:

Laboratory Exercise 1:
A. Acid Fast Stain: a few species in the bacteria genera Mycobacterium and Nocardia,
and the parasite Cryptosporidium, do not readily stain with simple stains. However,
these microorganisms can be stained by heating them with carbolfuchsin. The heat
drives the stain into the cells. Once the microorganisms have taken up the
carbolfuchsin, they are not easily decolorized by acid-fastness and are termed acidfast. This acid-fastness is due to the high lipid content (mycolic acid) in the cell
wall of these microorganisms. The Ziehl-Neelsen acid-fast staining procedure is a
very useful differential staining technique that makes use of his difference in
retention of carbolfuchsin. Acid-fast microorganisms will retained this dye and
appear red. Microorganisms that are not acid-fast, termed nod-acid fast, will appear
blue due to the counterstaining with methylene blue after they have been
decolorized by the acid-alcohol. A modification of this procedure that employs a
wetting agent (Tergitol No. 7) rather than heat to ensure stain penetration is known
as the Kinyoun staining procedure.
1. Kinyoun procedure:
a. Heat-fix the slide as previously directed.
b. Flood the slide for 5 minutes with carbolfuchsin prepared with
Tergitol No. 7 (heat is not necessary). Let it stain for 2 minutes.
c. Decolorize wit acid-alcohol and wash with tap water. Repeat this
step until no more color runs off the slide.
d. Counterstain with alkaline methylene blue for 2 minutes. Wash and
air dry. Do not blot.
e. Examine under oil. Acid-fast staining microorganisms stain red; the
background and other organisms stain blue.
B. KOH preparations: superficial fungal infections represent the colonization by
fungi of the outer surface of cutaneous structure. The presence of fungi can be
demonstrated in KOH preparations using skin scales, nail scrapings, or infected hair.
1. KOH examination procedures:
a. Using a dissecting needle, pick up several skin scales and place them
in a drop of 10% KOH.
b. Overlay with a clean coverglass and gently warm the wet preparation
over the flame of the Bunsen burner.
c. The slide is examined under low- and high-dry power objectives,
using much-reduced light, for the presence of short, convoluted
hyphae and/or arthroconidia. Fungal elements must be differentiated
from fibers of cotton, wool, and other fabrics as well as a mosaic of
cholesterol crystals and other artifacts.
C. India Ink: many bacteria have a slimy layer surrounding them, which are usually
referred to as capsule. The capsules composition, as well as its thickness, varies

with individual bacterial species. Often, a pathogenic bacterium with a thick capsule
will be more virulent than a strain with little or no capsule, since the capsule
protects the bacterium against the phagocytic activity of the hosts phagocytic cells.
India ink is used for negative staining of the capsule. Unstained cells surrounded by
a halo in a black background indicate the presence of a capsule. Examination of this
method must be done as soon as possible to avoid false positives due to drying of
the India ink.
1. Procedure:
a. Make a suspension of the organism in a drop of water on a clean
slide.
b. Put a drop of India ink next to it.
c. Carefully lower a coverslip over the two drops so that they mix
together. There should be a gradient in the concentration of the ink.
d. Examine under the microscope and find a field where you can see the
cells surrounded by a halo in a black background.
D. Lactophenol cotton blue (LPCN): LPCB is used both as a mounting fluid and as
stain for fungi. Lactic acid acts as a clearing agent and aids in preserving the fungal
structures; phenol acts as a killing agent; glycerol prevents drying; and cotton blue
gives color to the structures.
E. Gram Stain: probably the most widely used in bacteriology. Enables us to
differentiate between two bacterial cultures, which are morphologically
indistinguishable, yet of different species. The stain divides the bacteria into 2 large
groups:
1. Those that retain the crystal violet dye-iodine complex (violet) throughout
the staining procedure, termed gram-positive; and
2. Those that loose the crystal violet color complex upon decoloration with
95% alcohol and are stained red (counterstained), termed gram-negative.
3. Permeability theory: possible explanation to gram-staining. It proposes that
bacteria stain differently because of chemical and physical differences in
their cell walls.
a. Gram-positive cell walls; thick and chemically simple, composed
mainly of protein and cross-linked mucopeptides. Alcohol, according
to this theory, causes dehydration and shrinkage of the gram-positive
cell wall, thereby reducing the loss of substances such as crystal
violet.
b. Gram-negative cell wall: thin, complex, multi-layered structure
containing protein, mucopeptides, and lipids. When treated with
alcohol, the lipid dissolves and the primary stain is washed out.
c. The gram stain is only valid when done on young (less than 24 hours
old) cultures of bacteria.
d. Usually the first step in identifying bacteria.
4. Procedure:

a. Prepare slides for the Gram stain [ make three circles on the slide
place a small drop of saline or tap water on each circle and
thoroughly mix with the corresponding material allow the
preparations to dry, and fix them by passing them gently three times
through a Bunsen burner flame].
b. Stain the prepared slides with crystal violet for 1 minute, and then
wash with water for a few seconds. Drain the excess water.
c. Apply Grams iodine solution for 1 minute.
d. Decolorize with 95% alcohol until free color has been washed off
(~15 seconds). Wash slide with water and drain.
e. Counterstain smears for 30 seconds with Safranin, wash and allow
drying by placing the slide in a vertical position on blotting paper.
f. Observe under the microscope, using the oil immersion objective.
F. Lab demonstration:
Microorganism
Staphylococcus
aureus
Bacillus cereus
Moraxella
catarralis
Escherichia coli
Streptococcus
pyogenes

Gram Reaction

Bacterial
Morphology

Coccus

Bacillus

Coccus

Bacillus

Coccus

Laboratory Exercise 2:
A. Infectious material usually contains more than one kind of bacteria. All of them
should be studied individually and the first step is to separate each one as a pure
culture.
B. The most common methods for obtaining a pure culture are the pour plate and the
streak methods.
C. Pour plate method: a petri dish is employed because of its broad surface. The
solidifying substance used (agar) traps the individual organisms in place. Instead of
floating around when they multiply, as in a liquid medium, they produce a fixed
colony of organisms which grows to form a visible mass. If the original organisms
are trapped some distance apart, each visible organisms or clump of organism
develops into a separate, distinct colony.
D. Nutrient agar: gram positive and gram negative bacteria can grow in here.
Microbiological growth medium commonly used for the routine cultivation of nonfastidious bacteria. It is useful because it remains solid even at relatively high

temperatures. Also, bacteria grown in nutrient agar grows on the surface, and is
clearly visible as small colonies.
E. S-110: selective medium for Staphylococcus aureus because it contains salt.
F. MacConkey: selective medium for enteric Gram negative bacteria and differential
due to lactose fermentation. Lactose + = pink. Lactose - = uncolored.
Culture medium designed to grow Gram-negative bacteria and differentiate them
for lactose fermentation.
G. Streaking technique: for inoculating any type of agar plate. The surface of the agar
should be free of water from condensation before the streaking is done.
1. Place a loopful of the mixed broth culture near the periphery of the plate.
Flame the loop and allow it to cool.
2. With the flamed (sterilized) loop, spread the inoculum over the top quarter
of the plate.
3. Make one light sweep through the lower portion of this streaked area, turn
the plate at right angles and streak approximately one half of the remaining
portion, without overlapping the previous streaks.
4. Turn the plate a quarter turn and streak the remainder of the plate avoiding
streaked areas.
5. Incubate your petri-plate bottom-side up at 37C for 24 hours.

H.

Laboratory Exercise Results:

Agar Plate
Nutrient Agar
S-110
MacConkey

Colony Description
2 colonies: creamopaque and other
cream-translucid.
Colonies cream and
opaque
Pink colonies

Microscopic
Morphology
Opaque = coccus
Translucid = bacillus

Gram Reaction
Coccus = +
Bacillus = -

Coccus

Bacillus

I. Mixed Culture: cultures containing more than one type of microorganisms.

J. Pure Culture: cultures containing a single type of microorganism.
K. Aseptic technique: technique used to avoid contamination. Must be used to reduce
the likelihood of bacterial contamination. This usually involves disinfection of

working areas, minimizing possible access by bacteria from the air to exposed
media, and use of flames to kill bacteria which might enter vessels as they are
opened.
L. Selective medium: permits preferential growth of certain microorganisms.
M. Differential medium: distinguishes among different microorganisms by bringing
out variations in their reaction.