Bacterial Culture Media: classification,

types and uses

July 24, 2010 tankeshwar Bacteriology, Culture Media used in Microbiology, laboratory
diagnosis of Bacterial Disease, Microbiology 18
Culture media contains nutrients and physical growth parameters necessary for microbial growth. All
microorganisms cannot grow in a single culture medium and in fact many can’t grow in any known culture
medium.
Organisms that cannot grow in artificial culture medium are known as obligate parasites. Mycobacterium
leprae, rickettsias, Chlamydias, and Treponema pallidum are obligate parasites. Bacterial culture media can be
distinguished on the basis of composition, consistency and purpose.

Classification of culture media used in Microbiology

laboratory on the basis of consistency
1. Solid medium
solid medium contains agar at a concentration of 1.5-2.0% or some other, mostly inert solidifying agent.
Solid medium has physical structure and allows bacteria to grow in physically informative or useful
ways (e.g. as colonies or in streaks). Solid medium is useful for isolating bacteria or for determining
the colony characteristics of the isolate.
2. Semisolid media
They are prepared with agar at concentrations of 0.5% or less. They have soft custard like consistency
and are useful for the cultivation of microaerophilic bacteria or for determination of bacterial
motility.
3. Liquid (Broth) medium
These media contains specific amounts of nutrients but don’t have trace of gelling agents such as
gelatin or agar. Broth medium serves various purposes such as propagation of large number of
organisms, fermentation studies, and various other tests. e.g. sugar fermentation tests, MR-VR broth.

Classification of culture media based on the basis of composition

1. Synthetic or chemically defined medium
A chemically defined medium is one prepared from purified ingredients and therefore whose exact
composition is known.
2. Non synthetic or chemically undefined medium

Non-synthetic medium contains at least one component that is neither purified nor completely characterized
nor even completely consistent from batch to batch. Often these are partially digested proteins from various
organism sources. Nutrient broth, for example, is derived from cultures of yeasts.

Synthetic medium may be simple or complex depending up on the supplement incorporated in it. A simple
non-synthetic medium is capable of meeting the nutrient requirements of organisms requiring relatively few
growth factors where as complex non-synthetic medium support the growth of more fastidious
microorganisms.

Classification of Bacterial Culture Media based on the basis of purpose/

functional use/ application
Many special purpose media are needed to facilitate recognition, enumeration, and isolation of certain types of
bacteria. To meet these needs, numerous media are available.

1. General purpose media/ Basic media

Basal media are basically simple media that supports most non-fastidious bacteria. Peptone water, nutrient
broth and nutrient agar are considered as basal medium. These media are generally used for the primary
isolation of microorganisms.

Nutrient Agar

2. Enriched medium (Added growth factors):

Blood Agar

Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes them enriched
media. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Blood agar, chocolate
agar, Loeffler’s serum slope etc are few of the enriched media. Blood agar is prepared by adding 5-10% (by
volume) blood to a blood agar base. Chocolate agar is also known as heated blood agar or lysed blood agar.
3. Selective and enrichment media are designed to inhibit unwanted commensal or contaminating bacteria
and help to recover pathogen from a mixture of bacteria. While selective media are agar based, enrichment
media are liquid in consistency. Both these media serve the same purpose. Any agar media can be made
selective by addition of certain inhibitory agents that don’t affect the pathogen of interest. Various approaches
to make a medium selective include addition of antibiotics, dyes, chemicals, alteration of pH or a
combination of these.

a. Selective medium
Principle: Differential growth suppression
Selective medium is designed to suppress the growth of some microorganisms while allowing the growth of
others. Selective medium are agar based (solid) medium so that individual colonies may be isolated.

Examples of selective media include:

1. Thayer Martin Agar used to recover N.gonorrhoeae contains antibiotics; vancomycin, colistin and
nystatin.
2. Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contains 10% NaCl.
3. Potassium tellurite medium used to recover C.diphtheriae contains 0.04% potassium tellurite.

4. MacConkey’s Agar used for Enterobacteriaceae members contains bile salt that inhibits most gram
positive bacteria.
5. Pseudosel Agar (Cetrimide Agar) used to recover P. aeruginosa contains cetrimide (antiseptic agent).
6. Crystal Violet Blood Agar used to recover S. pyogenes contains 0.0002% crystal violet.
7. Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by incorporating
malachite green.
8. Wilson and Blair’s Agar for recovering S. typhi is rendered selective by the addition of dye brilliant
green.
9. Selective media such as TCBS Agar used for isolating V. cholerae from fecal specimens have elevated
pH (8.5-8.6), which inhibits most other bacteria.

b. Enrichment culture medium

Enrichment medium is used to increase the relative concentration of certain microorganisms in the culture
prior to plating on solid selective medium. Unlike selective media, enrichment culture is typically used
as broth medium. Enrichment media are liquid media that also serves to inhibit commensals in the clinical
specimen. Selenite F broth, tetrathionate broth and alkaline peptone water (APW) are used to recover
pathogens from fecal specimens.
4. Differential/ indicator medium: differential appearance:
Certain media are designed in such a way that different bacteria can be recognized on the basis of their colony
colour. Various approaches include incorporation of dyes, metabolic substrates etc, so that those bacteria that
utilize them appear as differently coloured colonies. Such media are called differential media or indicator
media. Differential media allow the growth of more than one microorganism of interest but with
morphologically distinguishable colonies.
Examples of differential media include:

1. Mannitol salts agar (mannitol fermentation = yellow)

2. Blood agar (various kinds of hemolysis i.e. α, β and γ hemolysis)
3. Mac Conkey agar (lactose fermenters, pink colonies whereas non- lactose fermenter produces pale or
colorless colonies.
4. TCBS (Vibrio cholerae produces yellow colonies due to fermentation of sucrose)

5. Transport media:
Clinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth of
contaminating organisms or commensals. This can be achieved by using transport media. Such media prevent
drying (desiccation) of specimen, maintain the pathogen to commensal ratio and inhibit overgrowth of
unwanted bacteria. Some of these media (Stuart’s & Amie’s) are semi-solid in consistency. Addition of
charcoal serves to neutralize inhibitory factors.

 Cary Blair transport medium and Venkatraman Ramakrishnan (VR) medium are used to transport
feces from suspected cholera patients.
 Sach’s buffered glycerol saline is used to transport feces from patients suspected to be suffering from
bacillary dysentery.
 Pike’s medium is used to transport streptococci from throat specimens.

6. Anaerobic media:
Anaerobic bacteria need special media for growth because they need low oxygen content, reduced oxidation –
reduction potential and extra nutrients.
Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin K. Such media may
also have to be reduced by physical or chemical means. Boiling the medium serves to expel any dissolved
oxygen. Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron
filings can render a medium reduced. Before use the medium must be boiled in water bath to expel any
dissolved oxygen and then sealed with sterile liquid paraffin.

Robertson Cooked Meat (RCM) medium that is commonly used to grow Clostridium spps contains a 2.5 cm
column of bullock heart meat and 15 ml of nutrient broth. Thioglycollate broth contains sodium thioglycollate,
glucose, cystine, yeast extract and casein hydrolysate.

Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated in the medium.
Under reduced condition, methylene blue is colorless.
7. Assay media
These media are used for the assay of vitamins, amino acids and antibiotics. E.g. antibiotic assay media are
used for determining antibiotic potency by the microbiological assay technique.
Other types of medium includes

 Media for enumeration of Bacteria,

 Media for characterization of Bacteria,
 Maintenance media etc.

Gram Staining: Principle, Procedure and

Results
February 2, 2015 tankeshwar Bacteriology, Staining techniques in Microbiology 42
Gram staining method, the most important procedure in Microbiology, was developed by Danish physician
Hans Christian Gram in 1884. Gram staining is still the cornerstone of bacterial identification and taxonomic
division.

This differential staining procedure separates most bacteria into two groups on the basis of cell wall
composition:

1. Gram positive bacteria (thick layer of peptidoglycan-90% of cell wall)- stains purple
2. Gram negative bacteria (thin layer of peptidoglycan-10% of cell wall and high lipid content) –stains
red/pink

Image 1: Basic classification of Medically

Important Bacteria

Nearly all clinically important bacteria can be detected/visualized using Gram staining method the only
exceptions being those organisms;

1. That exists almost exclusively within host cells i.e. Intracellular bacteria (e.g., Chlamydia)
 2. Those that lack a cell wall (e.g., Mycoplasma)
3. Those of insufficient dimensions to be resolved by light microscopy (e.g., Spirochetes)

Classic Gram staining techniques involves following steps:

1. Fixation of clinical materials to the surface of the microscope slide either by heating or by using
methanol. (# Methanol fixation preserves the morphology of host cells, as well as bacteria, and is
especially useful for examining bloody specimen material).

An easy way to remember the steps of the Gram stain

2. Application of the primary stain (crystal violet). Crystal violet stains all cells blue/purple
3. Application of mordant: The iodine solution (mordant) is added to form a crystal violet iodine (CV-I)
complex; all cells continue to appear blue.
4. Decolorization step: The decolorization step distinguishes gram-positive from gram-negative cells.
The organic solvent such as acetone or ethanol, extracts the blue dye complex from the lipid-rich, thin
walled gram negative bacteria to a greater degree than from the lipid poor, thick walled, gram-positive
bacteria. The gram negative bacteria appear colorless and gram positive bacteria remain blue.
5. Application of counter stain (safranin): The red dye safranin stains the decolorized gram-negative
cells red/pink; the gram-positive bacteria remain blue.

Principle of Gram Stain

Image 2: Cell wall of Gram Positive and Gram Negative Bacteria

The differences in cell wall composition of Gram positive and Gram negative bacteria accounts for the Gram
staining differences. Gram positive cell wall contain thick layer of peptidoglycan with numerous teichoic acid
cross linking which resists the decolorization.

In aqueous solutions crystal violet dissociates into CV+ and Cl – ions that penetrate through the wall and
membrane of both Gram-positive and Gram-negative cells. The CV+ interacts with negatively charged
components of bacterial cells, staining the cells purple.

When added, iodine (I- or I3-) interacts with CV+ to form large crystal violet iodine (CV-I) complexes within
the cytoplasm and outer layers of the cell.

The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes
of both gram-positive and gram negative bacteria.

The outer membrane of the Gram-negative cell (lipopolysaccharide layer) is lost from the cell, leaving the
peptidoglycan layer exposed. Gram-negative cells have thin layers of peptidoglycan, one to three layers deep
with a slightly different structure than the peptidoglycan of gram-positive cells. With ethanol treatment, gram-
negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell.
The highly cross-linked and multi-layered peptidoglycan of the gram-positive cell is dehydrated by the
addition of ethanol. The multi-layered nature of the peptidoglycan along with the dehydration from the
ethanol treatment traps the large CV-I complexes within the cell.

After decolorization, the gram-positive cell remains purple in color, whereas the gram-negative cell loses the
purple color and is only revealed when the counterstain, the positively charged dye safranin, is added.

Smear Preparation
Fix material on slide with methanol or heat. If slide is heat fixed, allow it to cool to the touch before applying
stain.

Image 3: Procedure of Gram Staining; note the color

change after each steps

Gram Staining Procedure/Protocol:

1. Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent. Please note
that the quality of the smear (too heavy or too light cell concentration) will affect the Gram Stain
results.
2. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
3. Flood slide with the mordant: Gram’s iodine. Wait 1 minute.
4. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
5. Flood slide with decolorizing agent (Acetone-alcohol decolorizer). Wait 10-15 seconds or add drop
by drop to slide until decolorizing agent running from the slide runs clear .
6. Flood slide with counterstain, safranin. Wait 30 seconds to 1 minute.
7. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then
blot dry with absorbent paper.
8. Observe the results of the staining procedure under oil immersion (100x) using a Bright field
microscope.
If you are struggling to remember the staining reagents used in this procedure and their order you can
remember this sentence “Come In And Stain” i.e. the order is Crystal violet, Iodine, Alcohol/Acetone and the
final one is Safranin.
Results:

 Gram-negative bacteria will stain pink/red and

 Gram-positive bacteria will stain blue/purple.

Reporting Gram smears

The report should include the following information:

Staphylococcus in Gram Stain

 Numbers of bacteria present, whether many, moderate, few, or scanty
 Gram reaction of the bacteria, whether Gram positive or Gram negative
 Morphology of the bacteria, whether cocci, diplococci, streptococci, rods, or coccobacilli.
Also, whether the organisms are intracellular.
 Presence and number of pus cells
 Presence of yeast cells and epithelial cells.

Example
An urethral smear report might read: ‘Moderate numbers Gram negative intracellular diplococci and many pus
cells.’

Quality Control: Always check new batches of stain and reagents for correct staining reactions using a
smear containing known Gram positive and Gram negative organisms.

Variations in Gram Staining Results

Various factors influence the results of Gram staining. Sometime the result might be entirely different than you
have anticipated. Find out about ‘variations in Gram Staining Results in this post’.