Gram staining and bacterial culture
Two of these techniques have been indispensible with regard to classical diagnostics, research, and
appreciation of the physiological states of microbes. In fact, the Danish bacteriologist referred to as Hans
Christian Gram is often credited with the development of Gram staining during the late 19th century.
Gram Staining :Hans Christian Gram invented the technique to differentiate the kind of bacteria based
on their cell wall appearance. He realized, in a series of staining steps, some bacteria retain a violet dye
and others lose that dye and take another color to lose the violet dye.
The first staining developed by Gram used crystal violet as the primary and iodine as the mordant, with
alcohol or acetone as a decolorizer and safranin as a counterstain. The staining technique revealed two
distinct groups of bacteria as no longer retaining the violet dye, for they reacted when subjected to being
decolorized with the alcohol or acetone—those which tested purple or blue once the decolorizer was
applied to them were classified as Gram-positive bacteria, while those which turned pink or red were
classified as Gram-negative bacteria.
Purpose of Gram Staining: Gram staining can thus be summarized as attempting to be the differential
staining approach suitable for a fast, illustrative characterization of the diagnostic bacterial isolate. Such
distinction by clinical microbiology between Gram-positive and Gram-negative bacteria guides
treatment, since the results of the Gram staining can be suggestive not only of the bacterial
pathogenicity but also of the clinical outcome of the treatment, bearing in mind the antibiotic sensitivity.
Gram Staining:
1. Preparation of Bacterial Smear:
Obtain a bacterial culture sample (e.g., from a patient specimen or a pure culture).
Place a small amount of the bacterial culture on a clean glass slide and spread it evenly
to create a thin bacterial smear.
2. Fixation:
Allow the bacterial smear to air dry completely.
Heat-fix the smear by passing it briefly through a flame (do not overheat, as it can
damage the bacterial cells).
3. Staining Process:
Flood the fixed bacterial smear with crystal violet stain (primary stain) and let it stand for
about 1 minute.
Rinse the slide gently with water to remove excess stain.
Flood the smear with iodine solution (mordant) and let it stand for another minute.
Iodine forms a complex with crystal violet, enhancing its retention in Gram-positive
bacteria.
4. Decolorization:
Rinse the slide with alcohol or acetone (decolorizer) for a few seconds. This step
removes the stain from Gram-negative bacteria but not from Gram-positive bacteria due
to their thicker peptidoglycan layer.
5. Counterstaining:
Counterstain the smear with safranin (secondary stain) for about 30 seconds. Safranin
stains Gram-negative bacteria pink or red, contrasting with the purple color of Gram-
positive bacteria.
6. Rinse and Dry:
Rinse the slide gently with water to remove excess stain and allow it to air dry.
7. Microscopic Examination:
Examine the stained bacterial smear under a light microscope using oil immersion
(1000x magnification).
Gram-positive bacteria will appear purple/blue, while Gram-negative bacteria will
appear pink/red.
Bacterial Culture:
1. Inoculation:
Obtain a sterile culture medium appropriate for the type of bacteria being cultured (e.g.,
nutrient agar, blood agar, MacConkey agar).
Using a sterile loop or swab, streak the bacterial culture sample onto the surface of the
agar medium in a pattern that allows for isolated colonies to develop (e.g., quadrant
streaking for pure culture isolation).
2. Incubation:
Place the inoculated agar plate in an incubator set at the optimal temperature and
atmospheric conditions for the growth of the bacteria being cultured (e.g., 37°C for most
human pathogens).
Incubate the plate for 24 to 48 hours, periodically checking for bacterial growth.
3. Observation and Colony Morphology:
After incubation, observe the agar plate for the presence of bacterial colonies.
Note the size, shape, color, texture, and other characteristics of the colonies, which can
provide preliminary information about the bacteria present.
4. Subculture and Identification:
If necessary, perform subcultures by transferring individual colonies from the primary
culture plate to new agar plates or broth media for further testing or identification.
Use biochemical tests, serological assays, molecular techniques (e.g., PCR), or other
methods to identify and characterize the bacterial species present.
Different types of agar used in bacterial culture provide specific nutrients and conditions to support the
growth of different types of bacteria and aid in their identification. One such agar is MacConkey agar,
which is commonly used to isolate and differentiate gram-negative bacteria, particularly those belonging
to the Enterobacteriaceae family.
MacConkey Agar: MacConkey agar was developed by Alfred Theodore MacConkey in the late 19th
century as a selective and differential medium for isolating and identifying gram-negative bacteria,
especially enteric pathogens. The composition of MacConkey agar includes:
1. Peptones and Proteose Peptones: Serve as a source of nitrogen and amino acids for bacterial
growth.
2. Lactose: Acts as the fermentable carbohydrate in the medium. Bacteria capable of fermenting
lactose produce acid, which lowers the pH of the agar, leading to color changes.
3. Bile Salts and Crystal Violet: Act as selective agents by inhibiting the growth of gram-positive
bacteria. Bile salts disrupt the cell membrane of gram-positive bacteria, making them susceptible
to inhibition.
4. Neutral Red: A pH indicator that turns pink/red in acidic conditions, indicating lactose
fermentation and acid production.
Purpose and Applications: MacConkey agar serves several purposes in microbiology laboratories:
1. Selective Medium: The presence of bile salts and crystal violet inhibits the growth of gram-
positive bacteria while allowing the growth of gram-negative bacteria.
2. Differential Medium: Based on their ability to ferment lactose, bacteria on MacConkey agar are
classified into lactose-fermenting (lactose-positive) and non-lactose-fermenting (lactose-
negative) colonies.
3. Identification of Enteric Pathogens: Enterobacteriaceae, such as Escherichia coli, Salmonella
spp., and Shigella spp., are commonly isolated and identified on MacConkey agar. Lactose-
fermenting colonies appear pink/red due to acid production, while non-lactose-fermenting
colonies remain colorless or pale.
Interpretation of Results:
Lactose-Fermenting (Lactose-Positive) Bacteria: These bacteria produce acid during lactose
fermentation, causing the agar around their colonies to turn pink/red (acidic).
Non-Lactose-Fermenting (Lactose-Negative) Bacteria: These bacteria do not ferment lactose
and do not produce acid. Their colonies remain colorless or pale on MacConkey agar.
Blood Agar:
Blood agar contains sheep's blood and provides nutrients for a wide range of bacteria. It is used to
differentiate bacteria based on their hemolytic properties:
Alpha-hemolysis: Incomplete hemolysis, resulting in a greenish discoloration around bacterial colonies.
Beta-hemolysis: Complete hemolysis, leading to a clear zone around colonies due to red blood cell lysis.
Gamma-hemolysis: No hemolysis, with no change in the appearance of the agar around colonies.
Chocolate Agar: Chocolate agar is enriched with heat-treated blood, providing nutrients for fastidious
bacteria, including Neisseria species and Haemophilus influenzae. It is used in diagnosing infections
caused by these organisms.
Sabouraud Agar: Sabouraud agar contains a low pH and high glucose concentration, making it selective
for fungi and inhibitory to most bacteria. It is commonly used for culturing and identifying
dermatophytes and yeast species.
Mannitol Salt Agar (MSA): MSA contains high salt concentrations and mannitol sugar, making it selective
for halophilic bacteria, particularly Staphylococcus aureus. Fermentation of mannitol by S. aureus leads
to acid production and a yellow color change in the agar.
Thayer-Martin Agar: Thayer-Martin agar is selective for Neisseria gonorrhoeae and Neisseria
meningitidis. It contains antibiotics (vancomycin, colistin, nystatin) to inhibit the growth of other bacteria
and yeast.
Cetrimide Agar: Cetrimide agar is selective for Pseudomonas aeruginosa, a common opportunistic
pathogen. It contains cetrimide, which inhibits the growth of many other bacteria, allowing for the
isolation and identification of P. aeruginosa.
Hektoen Enteric Agar (HEA): HEA is selective for enteric pathogens, including Salmonella and Shigella
species. It contains bile salts and dyes that inhibit the growth of non-enteric bacteria and differentiate
between lactose-fermenting and non-lactose-fermenting colonies.
Eosin Methylene Blue (EMB) Agar: EMB agar is selective for Gram-negative bacteria, particularly those
that ferment lactose. It contains eosin and methylene blue dyes, which inhibit the growth of Gram-
positive bacteria and differentiate between lactose-fermenting (dark colonies) and non-lactose-
fermenting (colorless colonies) bacteria.
