PHARMACEUTICAL MICROBIOLOGY LABORATORY WORKSHEET

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MICROBIOLOGICAL CULTURE MEDIA

The cultivation of microorganisms on an artificial growth medium requires that the medium supply
all the nutritional and energy requirements necessary for growth. However, in some cases, the specific
nutrient requirements for a certain organism to grow may not be known. In order to cultivate such an
organism, a medium using rich extracts of meat or plants that would supply all the amino acids,
nucleotide bases, vitamins, and other growth factors is used.

Inoculum refers to microorganisms introduced into a culture medium to initiate growth. The process
of transferring or isolating microorganisms from clinical specimen or from pure culture to a culture
medium is called inoculation. Culture or microbes that grow and multiply in or on a culture medium
maybe of several types:
1. Pure culture when only one species or one strain of microorganism is present
2. Mixed culture when there are two or more species or strain of microorganism
3. Contaminated culture when unwanted microorganisms are accidentally grown
4. Stock culture for research and school purposes

Classification of Culture Media A. According to Nutritional Composition

1. Simple or Basic Media consists only of the basic requirements for growing microorganisms.
It contains beef extract, peptone, and water. Beef extract provides vitamins, organic
growth factors, organic nitrogen, and carbon compounds needed to grow non-fastidious
microorganisms. Peptones are proteins with shorter chains of amino acids that can be
easily digested by bacteria.
Example: Nutrient agar, Nutrient broth, Peptone water
2. Complex Media refer to media where exact composition and amount of the individual
amino acids, vitamins, growth factors, and other components that make up the medium
are not exactly known. These may contain added substances that provide special nutrients.
Examples: Blood Agar Plate (BAP), Chocolate Agar Plate (CAP).
3. Synthetic Media or Defined Media have known chemical composition and the individual
components are weighed out exactly to make up the medium. It is a specially prepared
media for research purposes.
Example: Peptone water media (peptone water and 1% meat extract)

B. According to Physical State or Consistency

1. Solid Medium contains 1.5 to 3% agar. The visible growth of microorganism on solid media
is called colony.
Example: Nutrient agar (nutrient broth and 2% agar)
2. Liquid Medium has no agar. Growth of microorganisms is evidenced by turbidity, pellicle
formation and precipitation.
Examples: Nutrient broth (1% peptone, 0.5%NaCl and water)
Peptone water (peptone water and 1% meat extract)

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3. Semi-solid Medium contains 0.5% agar. It is used to demonstrate swarming growth of

Proteus and other motile organisms and as transport media.
Example: Sulfide Indole Motility (SIM) medium

C. According to Manner of Dispensing or Formation

1. Tubed Medium – dispensed in test tubes
a. Liquid
b. Semi-solid
c. Solid
i. Butt – inoculation is done through stabbing
ii. Slope or Slant – inoculation is done through streaking
iii. Butt-slant – inoculation is done through stabbing then streaking
2. Plated Medium – dispensed in Petri dishes or plates; inoculation is done through streaking

D. According to Function or Application (Refer to Table 1 for complete details)

1. Basal or Base Medium is one from which other culture media are prepared, i.e. Nutrient
Broth, Nutrient Agar, and Trypticase Soy Agar.
2. Enriched Medium contain additional requirements or additives for fastidious organisms to
grow. These additives may be blood, serum, or ascetic fluid. Only select microorganisms
produce hemolysis in sheep’s blood. Other blood sources can be horse or rabbit. Examples
are BAP, CAP, and Loeffler’s Serum Slant (LSS).
3. Selective Medium contain inhibitors to prevent growth of unwanted organisms and favor
desired organisms. These include media that:
a. Inhibit growth of Gram-positive organisms
i. EMB – Eosin Y and methylene blue
ii. SSA – bile salts and brilliant green
iii. MacConkey – bile salts and crystal violet
b. Inhibit growth of Gram-negative organisms
i. Cystine Tellurite Blood Agar (CTBA) – potassium tellurite
c. Inhibit swarming growth of Proteus
i. Phenylethyl alcohol agar (PEA) – alcohol
d. Inhibit contaminants or invaders
i. Antibiotics, i.e. penicillin and streptomycin

Indicators may also be added to demonstrate hydrogen sulfide production, carbohydrate

fermentation, and pH level.
a. Hydrogen sulfide indicator
i. Salmonella-Shigella Agar (SSA) and Xylose Lysine Deoxycholate (XLD) –
sodium thiosulfate
ii. Hektoen Entteric Agar (HEA) – ferric ammonium citrate
b. Carbohydrate fermentation
i. SSA – lactose ii. HEA
– sucrose, lactose, salicin

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4. Differential Medium contains dyes or indicators added to differentiate two groups of

organisms growing together.
a. Differentiates Staphylococcus spp. based on mannitol fermentation
i. Mannitol Salt Agar (MSA) – phenol red
b. Differentiates Salmonella from Shigella spp.
i. SSA – neutral red
c. Differentiates E. coli from other enterics
i. EMB – Eosin Y and methylene blue
5. Enrichment Medium is a liquid medium to which certain substances are added to enhance
the growth of pathogenic organisms and suppress other unwanted organisms
a. Enrichment medium for Salmonella typhi
i. Selenite broth
b. Solution enrichment medium for Vibrio cholera
i. Alkaline Peptone Water (APW)
6. Transport Medium is used when there is anticipated delay in bringing the specimen from
source to laboratory
a. Cary-Bair for Vibrio sp.
b. Stuart medium for Vibrio cholera
7. Assay Medium is used for research and assay of vitamins, amino acids and antibiotics
8. Special Medium for biochemical test
a. Tryptone broth for identifying Gram-negatives based on indole production
b. Methyl-red Voges-Proskauer (MR-VP) aids in the identification of enteric
gramnegative bacilli
c. Peptone Yeast Extract Glucose Broth is a non-selective, enriched medium that
facilitates the recovery of more fastidious microorganisms, such as Prevotella,
Porphyromonas, and the Bacteroides fragilis group, along with other obligately
anaerobic bacteria.
d. Starch Casein Agar (SCA) is a solid medium used for the detection of saccharolytic
marine bacteria. It is also used for the cultivation and enumeration of
Actinomycetes species from water and soil samples by the double-layer agar
technique.

Table 1. PLATING MEDIA FOR ROUTINE BACTERIOLOGY

MEDIUM COMPONENTS/COMMENTS PRIMARY PURPOSE

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BISMUTH Contains glucose as its fermentable sugar, Selective medium for

SULFITE AGAR brilliant green as inhibitor, bismuth sulfite as isolation and cultivation
(BSA) indicator and ferric sulfate as H2S indicator. of Salmonella spp, i.e.
Salmonella typhi

Color of Colony:
Metallic colonies with black ring
BLOOD AGAR Enriched and differential Cultivation of almost all
PLATES (BAP) Trypticase soy agar or beef heart infusion or bacteria, differential for
nutrient agar with 5% defibrinated sheep blood hemolytic organisms
Used in cultivation of
Colony Characteristics: fastidious
Gamma hemolysis (non-hemolysis) microorganisms based on
hemolytic reactions
• Streptococcal species do not lyse
(hemolyse) sheep’s RBC
• no discrete zones are formed around the
colony
Alpha hemolysis
• Streptococci modify hemoglobin to green
pigment (biliverdin and other heme
compounds)
• zone of partially lysed red cells surround
the colonies
• greenish discoloration
Beta hemolysis
• Streptococci create clear zone (complete
lysis of RBC)
CHOCOLATE AGAR Peptone base, enriched with solution of 1% Cultivation of
hemoglobin or Isovitalex (BBL) and Haemophilus spp. and
supplements pathogenic Neisseria spp.
COLISTINNALIDIXIC Columbia agar base with 10 mg colistin per Selective isolation of
ACID liter, 15 mg nalidixic acid per liter to inhibit gram-positive cocci
(CNA) AGAR Gramnegatives
EOSIN Selective and differential Isolation and
METHYLENE Peptone base with lactose (fermentable differentiation of
BLUE (EMB) carbohydrate) and sucrose. Eosin Y and lactosefermenting and
AGAR (LEVINE) methylene blue as pH indicators and inhibitors. nonlactose fermenting
Gramnegative enteric
Color of Colony bacilli Used to isolate
Lactose Fermentors - purple with green fecal coliforms
metallic sheen (Escherichia coli) Inhibits Gram positive
organisms

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Non-Lactose Fermentors - colorless (Shigella

and Salmonella)

MEDIUM COMPONENTS/COMMENTS PRIMARY PURPOSE

HEKTOEN Peptone base agar with bile salts as inhibitor, Differential, selective
ENTERIC AGAR lactose, sucrose and salicin as the fermentable medium for isolation and
(HEA) sugars, and ferric ammonium citrate and sodium differentiation of
thiosulfate as H2S indicators. pH indicator is Salmonella and Shigella
bromothymol blue. spp. from other
gramnegative enteric
bacilli
Color of Colony:
Lactose Fermentors - carrot orange/salmon
colored colonies with E. coli Non-Lactose
Fermentors
- green colonies with Shigella
- colonies with black center Salmonella
typhimurium
LOEFFLER’S Nutrient Agar with horse serum Used to cultivate
SERUM SLANT Corynebacterium
(LSS) diptheriae
MACCONKEY Peptone base with lactose fermentable Isolation and
AGAR carbohydrate). Gram-positive organisms differentiation of
inhibited by crystal violet and bile salts. Neutral lactosefermenting and
red as pH indicator (red at pH below 6.8 and nonlactose fermenting
colourless at pH greater than 6.8) enteric bacilli
Selective and differential
Color of Colony medium for Gramnegative
Lactose Fermentors - pink to red (E.coli, organisms
Klebsiella, Enterobacter)
Non-Lactose Fermentors - colorless
(Salmonella, Shigella, Proteus)

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MANNITOL SALT Peptone base, mannitol (fermentable sugar for Selective isolation of
AGAR (MSA) identification of most S. aureus strains), and staphylococci and
phenol red as indicator. Salt concentration micrococci
(NaCl) of 7.5% inhibits most bacteria Pathogenic staphylococci,
i.e. Staphylococcus aureus
Colonies capable of color change (from red to
yellow) of surrounding media as an indicator of
mannitol fermentation.
MODIFIED Blood agar base enriched with hemoglobin, Selective for Neisseria
THAYER-MARTIN growth factors, and antimicrobial agents; spp., i.e. N. gonorrhoeae
(MTM) AGAR contaminating organisms are inhibited by and N. meningitides
colistin, nystatin, vancomycin and trimethoprim
PHENYLETHYL Nutrient agar base. Phenyl ethanol inhibits Selective isolation of
ALCOHOL (PEA) growth of Gram-negative organisms by Gram-positive cocci
AGAR interfering with DNA synthesis (Staphylococcus and
Streptococcus), Bacillus,

MEDIUM COMPONENTS/COMMENTS PRIMARY PURPOSE

Clostridium, and
anaerobic Gram-
negative bacilli

Inhibits E. coli,
Salmonella sp., Shigella.
Enterobacter,
Pseudomonas
SALMONELLASHIGELLA Peptone base with lactose (fermentable Selective for Salmonella
AGAR carbohydrate), ferric citrate, and sodium and Shigella spp.
(SSA) citrate. Neutral red as pH indicator;
inhibition by brilliant green and bile salts,
and citrate.
Sodium thiosulfate and ferric citrate acts as
H2S indicator.

Color of Colony:
Lactose Fermentors – red (E. coli, Klebsiella,
Enterobacter)
Non-Lactose Fermentors - white with or
without black center
Salmonella- colorless with black center
Shigella- colorless

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THIOSULFATE Peptone base agar with yeast extract, bile Selective and
CITRATE-BILE salts, citrate, sucrose, ferric citrate, and differential for Vibrio
SALT SUCROSE sodium thiosulfate. spp.
(TCBS) AGAR Ox bile and sodium citrate acts as inhibitor,
and the sucrose is the fermentable
carbohydrate.
Bromthymol blue acts as pH indicator.
Sodium thiosulfate and ferric citrate acts as
the H2S indicator.

Color of Colony:
Yellow – V. cholera; Green – V.
parahemolyticus
XYLOSE LYSINE Yeast extract agar with xylose, lactose and Isolation and
DEOXYCHOLATE sucrose as fermentable carbohydrates, ferric differentiation of
(XLD) AGAR ammonium citrate and sodium thiosulfate as Salmonella and Shigella
H2S indicator. Sodium deoxycholate inhibits spp. from other
gram-positive organisms; phenol red as gramnegative enteric
indicator; bile salt as bacteriostatic agent. bacilli

Color of Colony:
Lactose Fermentors – yellow (E. coli)
Non-Lactose Fermentors – Red (Shigella)
- Red colonies with black center
(Salmonella)
COLONY CHARACTERISTICS OF REPRESENTATIVE MICROORGANISMS USED
FOR PHARMACEUTICAL TESTING IN PLATED MEDIA

Staphylococcus aureus Bacillus subtilis

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Escherichia coli Pseudomonas aeruginosa

Circular, moist, smooth and of entire Flat, small colony with mucoid

Round, smooth, raised, and glistening Flat, white, circular lobate, irregular
with gray to deep golden yellow color colonies
margin with flat and pink appearance appearance
(MacConkey agar)

Candida albicans Aspergillus brasiliensis

White to cream-colored, smooth, White to yellow and turning black with

glabrous, and yeast-like colonies cottony appearance
(Sabouraud Dextrose Agar)

EXERCISE 3 MICROBIOLOGICAL CULTURE MEDIA

OBJECTIVE:
Demonstrate understanding of basic laboratory skills in the preparation of different forms of culture
media for use in microbiological applications

MATERIALS:
Erlenmeyer flask Nutrient Agar
Beaker Spore strip/Indicator label
Spatula Wasserman test tubes
Graduated cylinder Loeffler’s test tubes

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Gauze Screw-capped tubes

Aluminum foil Kahn test tubes

GENERAL PROCEDURE:
A. PREPARING A SOLID PLATED MEDIA
1. Compute for the total batch size to be prepared. Each plate is estimated to contain 20 mL
medium. A 40 mL excess per batch is commonly included to compensate of losses.
2. Determine the amount of dehydrated medium needed for the computed batch.
3. Weigh needed dehydrated medium in an Erlenmeyer flask.
4. Add required volume of water with constant stirring and gentle heating until the culture
medium becomes clear and straw-colored.
5. Plug the Erlenmeyer flask with gauze and cover with aluminum foil. Secure the foil using
masking tape. Label the flask. Stick a spore strip in the label.
6. Sterilize by autoclaving at 121oC for 15 minutes at 15 psi.
7. Dispense c. 20 mL per Petri dish.
8. Allow the plated culture medium to solidify on a flat surface. Petri dish can be partially
opened to allow some moisture to escape.
9. After the culture solidifies, cover the Petri dish and label (gum label) with the name of the
culture medium.
10. Wrap in aluminum foil secured with masking tape. Label.
11. Place inside the biological refrigerator in inverted position.

B. PREPARING A SOLID TUBED MEDIA

1. Compute for the total amount of batch to be prepared. Each butt-slant requires about
7 mL slant contains about 5 mL, and deep/butt tubes need about 3 mL media. A 50 mL
excess is commonly included to compensate for compounding losses.
2. Calculate the dehydrated medium needed for the computed batch.
3. Weigh needed dehydrated medium in an Erlenmeyer flask.
4. Add required volume of water with constant stirring and gentle heating until the culture
medium becomes clear and straw-colored.
5. Dispense 7 mL in screw-capped test tubes, 5 mL in Loeffler’s test tube, and 3 mL in
Wasserman test tube.
6. Plug the Loeffler’s and Wasserman test tubes with cotton.
7. Place the test tubes in a beaker, then cover with aluminum foil secured with masking tape.
Label with the name of culture media.
8. Sterilize by autoclaving at 121oC for 15 minutes at 15 psi.
9. Allow the medium to solidify as intended. Screw-capped tubes (7 mL) to form with
buttslant, Loeffler’s test tube (5 mL) to form with slant, and Wasserman test tube (3 mL)
to form with deep/butt culture media.
10. Once the media solidifies, label each test tube with the name of the culture media.
11. Place in a beaker, wrap in aluminum foil secured with masking tape. Label.
12. Place inside the biological refrigerator.

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C. PREPARING A LIQUID MEDIA

1. Compute for the total amount of batch to be prepared. Each culture media broths requires
estimated 2 mL to be placed in Kahn test tubes. A 10 mL excess is commonly added to
compensate for compounding losses.
2. Calculate the dehydrated medium needed for the computed batch.
3. Weigh needed dehydrated medium in an Erlenmeyer flask.
4. Add required volume of water with constant stirring and gentle heating until the culture
medium becomes clear and straw-colored.
5. Dispense 2 mL in Kahn test tubes.
6. Plug with cotton. Place the test tubes in a beaker, then cover with aluminum foil secured
with masking tape. Label with the name of culture media.
7. Sterilize by autoclaving at 121oC for 15 minutes at 15 psi.
8. Allow to cool then place inside the biological refrigerator.

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MICROSCOPY WITH OIL IMMERSION - Oil immersion microscopy is

essential to any microbiology laboratory. When light passes from a material of one
refractive index to material of another, as from glass to air or from air to glass, it
bends. Light of different wavelengths bends at different angles, so that as objects
are magnified the images become less and less distinct. With dry objective lenses,
the loss of resolution prevents using magnifications of above 400x or so. In fact,
even at 400x the images of very small objects are badly distorted. Placing a drop of
oil with the same refractive index as glass between the cover slip and objective lens
eliminates two refractive surfaces, so that magnifications of 1000x or greater can be
achieved while still preserving good resolution.

The objective lens must be designed specifically for oil immersion microscopy. The
oil immersion lens should be used on fixed (dead - not moving) specimen that is no
thicker than a few micrometers, and specifically when the structures to be viewed
are quite small (one or two micrometers in dimension). Oil immersion is essential in
viewing individual bacteria or details of the striations of skeletal muscle. It is nearly
impossible to view living, motile protists at a magnification of 1000x, except for the
very smallest and slowest.

Cedar Wood oil was the immersion oil of choice for many years before the large
scale manufacture of synthetic alternatives. However, this oil can have many
disadvantages. If not correctly cleaned up after use, it can penetrate and damage the
cement which holds the objective front lens in place. Cedarwood oil can also turn
yellow with age and has a tendency to absorb light in the ultraviolet and blue range
of the spectrum.

Modern synthetic oils are designed to remain colour stable over time and are
relatively inert. Most oils are designed to work at room temperature (i.e., 23°C). A
change in temperature causes a change in the refractive index of the oil. A
temperature difference of only 1°C can cause a change in the refractive index of the
oil by a factor of 0.0004. When capturing images over many hours, these subtle
differences will be present in the images and data collected. If carrying out long-
term, live-cell imaging experiments that require temperature-controlled chambers
around the cells and stage, use commercial oil designed to work at 37°C.

Immersion oils can (and will) penetrate the microscope components and can
damage dry objectives, corroding the cement used to hold objective front lenses in
place. To clean the immersion objective use a lens cleaning tissue to sweep across

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the surface of the objective front lens in one direction only. Continue cleaning in the
same manner (using a clean section of lens tissue for each sweep) until no oil is
seen on the tissue. A commercial immersion oil removal solutions or a small
amount of xylene may also be used for the final cleaning.

STAINING
A bacterial smear is a small amount of culture spread in a very thin film on the
surface of the slide. To prevent the bacteria from washing away during the staining
steps, the smear may be chemically or physically “fixed” to the surface of the slide.
Heat fixing is an easy and efficient method, and is accomplished by passing the
slide briefly through the flame of a Bunsen burner, which causes the biological
material to become more or less permanently affixed to the glass surface. Heat fixed
smears are ready for staining. In a simple stain, dyes that are either attracted by
charge (a cationic dye such as methylene blue or crystal violet) or repelled by
charge (an anionic dye such as eosin or India ink) are added to the smear. Cationic
dyes bind the bacterial cells which can be easily observed against the bright
background. Anionic dyes are repelled by the cells, and therefore the cells are bright
against the stained background.

SIMPLE STAIN
In simple staining, a single dye is used to emphasize particular structures in the
specimen. A simple stain will generally make all of the organisms in a sample
appear to be the same color, even if the sample contains more than one type of
organism. Simple staining can be used for all types of bacterial cells to give contrast
to the otherwise colorless cell in order to determine cell morphology, size, and cell
grouping. This technique is simple because only one dye is used and direct, because
the actual cell is stained.

Methylene blue, crystal violet, and Ziehl’s carbolfuschin are basic dyes, and
therefore work on the same principle; however, crystal violet is the darkest dye and,
thus, is most easily viewed through the microscope and gives the best contrast to the
unstained background. Another advantage of crystal violet is that most laboratories
that work with bacteria will have this stain readily available due to its use in the
Gram stain. CAUTION: Dyes used for bacteriological staining are usually aniline
dyes so they are potentially carcinogenic and should be handled carefully. Avoid
contact, inhalation, or ingestion of dye.

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DIFFERENTIAL STAINING TECHNIQUES

In microbiology, differential staining techniques are used more often than simple
stains as a means of gathering information about bacteria. Differential staining
methods, which typically require more than one stain and several steps, are referred
to as such because they permit the differentiation of cell types or cell structures. The
most important of these is the Gram stain. Other differential staining methods
include the endospore stain (to identify endospore-forming bacteria), the acid-fast
stain (to discriminate Mycobacterium species from other bacteria), a metachromatic
stain to identify phosphate storage granules, and the capsule stain (to identify
encapsulated bacteria).

GRAM STAIN
The gram stain ranks as one of the most important stains for bacteria. Named after
Hans Christian Gram who developed the method in 1884, the Gram stain allows
one to distinguish between Gram-positive and Gram-negative bacteria on the basis
of differential staining with a crystal violet–iodine complex and a safranin
counterstain. The cell walls of Gram-positive organisms retain this complex after
treatment with alcohol and appear purple. Gram negative cell walls have an outer
membrane (also called the envelope) that dissolves during the alcohol wash. This
permits the crystal violet dye to escape. Only the decolorized cells take up the pink
dye safranin, which explains the difference in color between the two types of cells.
At the conclusion of the Gram stain procedure, Gram positive cells appear purple,
and Gram negative cells appear pink. The method described is useful for assessing
bacterial contamination of tissue culture samples, or for examining the Gram stain
status and morphological features of bacteria isolated from mixed or isolated
bacterial cultures.

ACID FAST STAIN

Some bacteria produce the waxy substance mycolic acid when they construct their
cell walls. Mycolic acid acts as a barrier, protecting the cells from dehydrating, as
well as from phagocytosis by immune system cells in a host. This waxy barrier also
prevents stains from penetrating the cell, which is why the Gram stain does not
work with mycobacteria such as Mycobacterium, which are pathogens of humans
and animals. For these bacteria, the acid–fast staining technique is used.

To perform the acid-fast stain, a heat-fixed smear is flooded with the primary stain
carbolfuchsin, while the slide is heated over a steaming water bath. The heat
“melts” the waxy cell wall and permits the absorption of the dye by the cells. The
slide is allowed to cool, and a solution of acid and alcohol is added as a decolorizer.

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Cells that are “acid-fast” because of the mycolic acid in their cell wall resist
decolorization and retain the primary stain. All other cell types will be decolorized.
Methylene blue is then used as a counterstain. In the end, acid-fast bacteria (AFB)
will be stained a bright pink color, and all other cell types will appear blue.

STAINING METHODS TO HIGHLIGHT SPECIFIC CELL STRUCTURES

Capsule: The polysaccharide goo that surrounds some species of bacteria and a few
types of eukaryotic microbes is best visualized when the cells are negative stained.
In this method, the bacteria are first mixed with the stain, and then a drop of the
mixture is spread across the surface of a slide in the thin film. With this method,
capsules appear as a clear layer around the bacterial cells, with the background
stained dark.

Metachromatic granules or other intracytoplasmic bodies: Some bacteria may

contain storage bodies that can be stained. One example is the Gram positive bacilli,
Corynebacterium, which stores phosphate in structures called “volutin” or
metachromatic granules that are housed within the cell membrane. Various staining
methods are used to visualize intracytoplasmic bodies in bacteria, which often
provide an identification clue when observed in cells.

Endospore Stain: Endospores are dormant forms of living bacteria and should not
be confused with reproductive spores produced by fungi. These structures are
produced by a few genera of Gram-positive bacteria, almost all bacilli, in response
to adverse environmental conditions. Two common bacteria that produce
endospores are Bacillus or Clostridium. Both live primarily in soil and as symbionts
of plants and animals, and produce endospores to survive in an environment that
change rapidly and often. Mature endospores are highly resistant to environmental
conditions such as heat and chemicals and this permits survival of the bacterial
species for very long periods.

The process of endosporulation (the formation of endospores) involves several

stages. After the bacterial cell replicates its DNA, layers of peptidoglycan and
protein are produced to surround the genetic material. Once fully formed, the
endospore is released from the cell and may sit dormant for days, weeks, or years.
When more favorable environmental conditions prevail, endospores germinate and
return to active duty as vegetative cells.

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Because the endospore coat is highly resistant to staining, a special method was
developed to make them easier to see with a brightfield microscope. This method,
called the endospore stain, uses either heat or long exposure time to entice the
endospores to take up the primary stain, usually a water soluble dye such as
malachite green since endospores are permeable to water. Following a
decolorization step which removes the dye from the vegetative cells in the smear,
the counterstain safranin is applied to provide color and contrast. When stained by
this method, the endospores are green, and the vegetative cells stain pink.

Although endospores themselves are resistant to the Gram stain technique, bacterial
cells captured in the process of creating these structures can be stained. In this case,
the endospores are seen as clear oval or spherical areas within the stained cell.
Endospores can also be directly observed in cells by using phase contrast
microscopy

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