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Culture Media and Methods, Identification CHAPTE R

Chapter
of Bacteria by Conventional, Automated Text
and Molecular Methods
3
1.3
CULTURE MEDIA
The basic constituents of culture media are:
• Peptone: Mixture of partially digested proteins
• Agar: It is used for solidifying the culture media. It has no nutritional property.
○ It is prepared from seaweeds (red algae of species Gelidium and Gracilaria).
○ Agar is preferred over gelatine, as it is bacteriologically inert, and it melts at 98°C and
usually solidifies at 42 C
Concentration of agar: ○ Concentration of agar:
• For solid agar preparation For solid agar: 1–2% (Japanese agar 2% or New Zealand agar 1.2%)
1–2% (Japanese agar 2% or For semisolid agar: 0.5%
New Zealand agar 1.2%)
• For semisolid agar 0.5%
For solid agar to inhibit Proteus swarming: 6%
• For solid agar to inhibit • Others: Meat extract, Yeast extract, Blood and serum, Water and Electrolytes (NaCl).
Proteus swarming 6%
Simple/Basal Media
They contain minimum ingredients that support the growth of non-fastidious bacteria.
Examples include:
1. Peptone water: It contains peptone (1%) NaCl (0.5%) water
2. Nutrient broth: It is made-up of peptone water meat extract (1%)
3. Nutrient agar: It is made-up of nutrient broth 2% agar
The basal media are used for:
Simple/Basal Media: • Testing the non-fastidiousness of bacteria
• Peptone water: It contains • They serve as the base for the preparation of many other media
peptone (1%) + NaCl (0.5%) • Nutrient broth is used for studying the bacterial growth curve
+ water
• Nutrient agar is the preferred medium for:
• Nutrient broth: It is made up of
peptone water + meat extract ○ Performing the biochemical tests such as oxidase, catalase and slide agglutination
(1%). ○ To study the colony character and Pigment demonstration.
• Nutrient agar: It is made up of
nutrient broth + 2% agar Enriched Media
When a basal medium is added with additional nutrients such as blood, serum or egg, it is
called enriched medium. They also support the growth of fastidious bacteria, e.g.:
1. Blood agar: Prepared by adding 5–10% of sheep blood to the molten nutrient agar at
45 C. It is used to test the hemolytic property of the bacteria
Differential Media: 2. Chocolate agar: It is the heated blood agar, blood is added to the molten nutrient agar at
• MacConkey aAgar 70°C. It is more nutritious than blood agar, and even supports Haemophilus influenzae.
• CLED agar 3. oef er s serum slope is used for isolation of Corynebacterium diphtheriae.
4. Blood culture media: Used for blood culture. They are of two types:
○ Monophasic medium is made-up of brain heart infusion (BHI) broth
○ Biphasic medium has a liquid phase (BHI broth) and a solid agar slope (BHI agar).

Enrichment Broth
Liquid media that allow certain organism (pathogens) to grow and inhibit others (normal flora):
• Selenite F and Tetrathionate broth used for Salmonella and Shigella
• Alkaline peptone water (APW) used for Vibrio cholerae.

Selective Media
Solid media that allow certain organism (pathogens) to grow and inhibit others (normal flora):
1. Lowenstein Jensen (LJ) medium is used for isolation of Mycobacterium tuberculosis

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Manila Adventist College
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Chapter 3 - Culture Media and Methods, Identification of
Bacteria by Conventional, Automated and Molecular Methods

Culture Media and Methods, Identification of Bacteria by Conventional, Automated and Molecular Methods 33

2. Thiosulphate Citrate Bilesalt Sucrose (TCBS) agar used for isolation of Vibrio species
3. For the isolation of enteric pathogens such as Salmonella and Shigella from stool:
• DCA (Deoxycholate Citrate Agar)
• XLD (Xylose Lysine Deoxycholate) agar
4. Potassium tellurite agar (PTA) is used for isolation of Corynebacterium diphtheriae.
5. Wilson Blair bismuth sulphite medium: It is used for isolation of Salmonella Typhi.

Transport Media
They are used for the transport of specimens containing delicate organism or when the delay
is expected. Bacteria do not multiply in the transport media, they only remain viable.

Table 1.3.1: Transport media used for common bacteria

Organism Transport media
Streptococcus Pike’s medium
Neisseria Amies medium, Stuart’s medium
Vibrio cholerae VR (Venkatraman-Ramakrishnan) medium, Cary Blair medium and Autoclaved
sea water
Shigella, Salmonella Buffered glycerol saline, and Cary Blair medium

Differential Media
These media differentiate between two groups of bacteria by using an indicator.
1. MacConkey agar: It differentiates organisms into LF or lactose fermenters (produce
pink colonies, e.g. E. coli and Klebsiella) and NLF (produce colorless colonies, e.g. Shigella
and Salmonella).
2. CLED agar (Cysteine lactose electrolyte-deficient agar): This is similar to MacConkey
agar, differentiates between LF and NLF. It is used as an alternative to combination of
blood agar and MacConkey agar, for the processing of urine specimens:
• Advantages over MacConkey agar: It is less inhibitory than MacConkey agar,
supports gram-positive bacteria (except hemolytic Streptococcus) and Candida.
• Advantage over blood agar: It can prevent the swarming of Proteus.

Anaerobic Culture Media

Anaerobic media contain reducing substances which take-up oxygen and create lower redox
potential and thus permit the growth of obligate anaerobes such as Clostridium. Examples are:
1. Robertson s cooked meat (RCM) broth: It contains chopped meat particles (beef General Microbiology
heart), which provide glutathione and unsaturated fatty acids.
2. ther anaerobic media include:
• BHIS agar: Brain heart infusion agar with supplements (vitamin K and hemin)
• Thioglycollate broth, Anaerobic blood agar
• Neomycin blood agar, Egg yolk agar and Phenylethyl agar.

CULTURE METHODS
Various Aerobic Culture Methods
1. Streak culture by using loop with intermittent heating: It is the routinely used method.
2. Lawn or carpet culture: Useful for Carrying out antimicrobial susceptibility testing by
disk diffusion method, Bacteriophage typing and producing large amount of bacterial
growth for preparation of bacterial antigens and vaccines.
3. Stroke culture (zigzag fashion by straight wire): Used for citrate, urease and TSI (triple
sugar iron test)

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34 Review of Microbiology and Immunology

4. Stab culture: Stabbing the semisolid agar butt by a straight wire. It is used for:
(i) maintaining stock cultures, (ii) OF test (iii) Mannitol motility medium, (iv) Nutrient
agar semisolid butts, (v) TSI (here both stroke and stab cultures are made).
5. Liquid culture:
• Uses: Liquid cultures are useful for: (i) blood culture, (ii) for sterility testing.
• Advantages: Used for: (i) When bacteria load is less, (ii) specimens (e.g. blood)
containing antibiotics it is neutralized by dilution in the medium, (iii) when large
Aerobic culture methods: yields of bacteria are required, (iv) for demonstration of bacterial growth curve
• Streak culture • Disadvantages: Liquid cultures do not provide a pure culture from a mixed inoculum.
• Lawn or carpet culture
• Stroke culture
6. Pour-plate culture: Quantitative culture method, used to estimate viable bacterial count.
• Stab culture
• Liquid culture Incubatory Conditions
• Pour-plate culture • Most of the pathogenic organisms grow best at 37°C.
• Candle jar: It provides capnophilic atmosphere (3–5% CO2). This is useful for Brucella
abortus, Streptococcus, pneumococcus and gonococcus.
• Microaerophilic bacteria such as Campylobacter and Helicobacter require 5% oxygen.

Anaerobic Culture Methods

1. Production of vacuum
2. By displacement and combustion of oxygen: This principle is used in:
Anaerobic Culture Methods: • McIntosh and Filde’s anaerobic jar (It involves evacuation of air and replacement
• Production of vacuum
with hydrogen gas manually)
• By displacement and
combustion of oxygen: • Anoxomat System (principle is same, but done by automated instrument)
○ McIntosh and Filde's an- 3. Gas pak System (Absorption of oxygen chemically, e.g. using alkaline pyrogallol).
aerobic jar Indicators of anaerobiosis are:
○ Anoxomat System
• Chemical indicator: Reduced methylene blue
• Gas pak System
• Anaerobic Glove Box • Biological indicator: Pseudomonas
• By reducing agents 4. Anaerobic Glove Box (or anaerobic chamber)
• PRAS media 5. By reducing agents: Such as glucose, thioglycollate, meat, cysteine and ascorbic acid.
6. PRAS media (Prereduced, Anaerobically Sterilized).

Preservation of Microorganisms
• Short-term methods: (i) Sub-culturing, (ii) Immersing the culture in glycerol, or sterile
distilled water, (iii) Freezing at –20 C and (iv) Drying (for moulds and spores).
• ong-term methods: By Ultra temperature freezing and Lyophilization (freeze-drying).

IDENTIFICATION OF BACTERIA
Identification of bacteria can be done by: (i) conventional (culture and identification by
biochemical reactions), (ii) automated culture techniques (iii) molecular methods.

Automated Culture Techniques

Section 1

Conventional culture methods often yield poor results because of low bacterial load.
Therefore, various automated blood culture techniques have been in use since last decade.
Advantages: The major advantages of automated blood culture techniques are:
• Continuous automated monitoring (once in every 15–20 min by the instrument).
• Other advantages: More sensitive, yield, rapid, less labor intensive
Disadvantages: (i) high cost (ii) inability to observe the colony morphology as liquid medium
is used, (iii) no separate detection in mixed cultures, (iv) radioactive hazards for BACTEC.

MOLECULAR METHODS
Nucleic acid amplification techniques (NAATs) include:
• Polymerase chain reaction (PCR) and its modification including Real time PCR
• Ligase chain reaction (LCR) and Transcription-mediated amplification (TMA)

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Chapter 3 - Culture Media and Methods, Identification of
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Culture Media and Methods, Identification of Bacteria by Conventional, Automated and Molecular Methods 35

Table 1.3.2: Automated culture systems in diagnostic bacteriology

For bacterial culture Principle used
1. BACTEC In was radiometry based, but later changed to fluorescent based detection technique
2. BacT/Alert CO2 liberated from bacteria, causes a pH change, detected by colorimetry
3. ESP culture system CO2 liberated from bacteria, causes a pressure change, detected by manometry
For bacterial identification • Phoenix bacterial identification system
• MALDI –TOF (Matrix-assisted laser desorption ionization time-of-flight), e.g. VITEK MS
• VITEK 2 bacterial identification and antimicrobial sensitivity system
• Microscan Walkaway system
For M. tuberculosis MGIT (Mycobacterial Growth Indicator Tube)

• Nucleic acid sequence based amplification (NASBA)

• Strand displacement amplification (SDA).

Polymerase Chain Reaction (PCR)

PCR is a technology in molecular biology used to amplify a single or few copies of a piece of
DNA to generate millions of copies of DNA. It was developed by Kary B Mullis. PCR involves three basic steps:
• DNA extraction from the
Principle: PCR involves three basic steps. organism
1. DNA extraction from the organism • Amplification of extracted DNA
• Gel electrophoresis of
2. Ampli cation of e tracted A: The extracted DNA is subjected to repeated cycles amplified product
(30–35 numbers) of amplification in a thermocycler which takes about 3–4 hours. Each
amplification cycle has three steps:
• Denaturation at 95°C: This involves separation the dsDNA into two separate ssDNA.
• Primer annealing (55°C): Primer is a short oligonucleotide complementary to a small
sequence of the target DNA. It anneals to the complementary site on ssDNA.
• Extension of the primer (72°C): This step is catalyzed by Taq Polymerase enzyme. Each amplification cycle has
3. Gel electrophoresis of amplified product: The amplified DNA is electrophoretically three steps:
• Denaturation at 95°C
migrated according to their molecular size to form bands; seen under UV rays. • Primer annealing (55°C)
Advantages: PCR has the following advantages compared to the conventional culture: • Extension of the primer
• More sensitive: It can amplify very few copies of a specific DNA, so it is more sensitive. (72°C)
• More specific: By use of primers targeting specific DNA sequence of the organism
• Detects the organism: (i) either from sample, (ii) to confirm culture isolate.
• Detect the organisms that are highly fastidiousor noncultivable by conventional culture
methods.
• Detect genes coding drug resistance (e.g. MecA gene detection in Staphylococcus aureus)
• Detects genetic diseases such as sickle cell anemia, phenylketonuria, etc.
General Microbiology
Disadvantages:
• Conventional PCR detects only the DNA, but not the RNA (detected by RT-PCR).
• Qualitative, not quantitative (Quantitation is done by real time PCR).
• Viability: PCR cannot differentiate between viable or nonviable organisms.
• False positive amplification may occur due to contamination with environmental DNA.
• False negative: by PCR inhibitors present in some specimens such as blood, feces, etc.

Modification of PCR
1. Reverse transcriptase PCR (RT-PCR): For amplifying RNA, RT-PCR is done.
• After RNA extraction, the first step is addition of reverse transcriptase enzyme that
coverts RNA into DNA. Then, the amplification of DNA and gel documentation
steps are similar to that described for conventional PCR. Modification of PCR:
• Useful for detection of RNA viruses or 16SrRNA genes of the organisms. • Reverse transcriptase PCR
2. Nested PCR: The amplified products of the first round PCR is subjected to another round • Nested PCR
• Multiplex PCR
of amplification using a second primer targeting a different gene of same organism.

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Chapter 3 - Culture Media and Methods, Identification of
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36 Review of Microbiology and Immunology

• More sensitive: Double round of amplification yields high quantity of DNA.

• More specific: Use of two primers targeting same organisms makes more specific.
Bacteriophage typing is done • Disadvantage: There is more chance of contamination of the PCR tubes, which may
for: lead to false positive results.
• Staphylococcus aureus 4. Multiplex PCR: It uses more than one primer which can detect many DNA sequences of
• SalmonellaTyphi several organisms in one reaction.
• Vibrio cholerae
• Brucella • Syndromic approach: To diagnose infectious syndrome caused by more than one
• Corynebacterium diphtheriae organism.
• Contamination chances of reaction tubes with environmental DNA.

Real-time PCR (rt-PCR)

Real time PCR, though expensive, but has many advantages over a conventional PCR:
• Quantitative, hence can be used for monitoring treatment response, e.g. in HIV or HBV.
• Takes less time: As the amplification can be visualized even when the amplification cycle
is going on.
Bacteriocin typing is done for: • Contamination rate is extremely less.
• Shigella sonnei (colicin typing)
• Klebsiella (klebocin typing), • Sensitivity and specificity of rt-PCR assays are extremely higher.
• E.coli (colicin typing)
• Proteus (proticin typing), Detection of amplification products of real time PCR
• Pseudomonas (pyocin typing) • Nonspecific methods use SYBR green dye that stains any nucleic acid nonspecifically.
• Specific methods use fluorescent labelled DNA probe such as (i) TaqMan or hydrolysis
probe, (ii) Molecular beacon and (iii) FRET (Fluorescence Resonance Energy Transfer)
probe.

Biotyping is done for: MICROBIAL TYPING

• C. diphtheriae
• Vibrio cholerae Microbial typing refers to characterization of an organism beyond its species level. It is used
to determine the relatedness between different microbial strains of the same species and
thereby helps to (i) investigate outbreaks, (ii) determine the source and routes of infections.
Genotypic methods are more reliable and have better reproducibility and discriminative
power than phenotypic methods, however they are expensive.

Table 1.3.3: Typing methods

Phenotypic methods Genotyping methods
Bacteriophage typing: Based on their susceptibility to bacteriophages. It is done for on Amplification based methods
• Staphylococcus aureus and SalmonellaTyphi 1. Plasmid profile analysis
• Vibrio cholerae, Brucella and Corynebacterium diphtheriae 2. Chromosomal DNA analysis
Bacteriocin typing: Based on the ability of a strain to produce particular bacteriocin which 3. RFLP (Restricted fragment length
inhibits the growth of a set of selected indicator strains. It is done for: polymorphism)
• Shigella sonnei (colicin typing) 4. Ribotyping (RFLP analysis of
Section 1

• Klebsiella (klebocin typing), E.coli (colicin typing) ribosomal DNA)

• Proteus (proticin typing),Pseudomonas (pyocin typing) 5. Pulse field gel electrophoresis
(PFGE)-Gold standard method
Biotyping: Based on different biochemical properties of the organism. It is done for Amplification based methods
• C.diphtheriae (gravis, intermedius and mitis 1. PCR-RFLP
• Vibrio choleraeO1(classical and El Tor) and Yersinia pestis 2. Amplified Fragment Length
Antibiogram typing: Based on their resistance pattern to different antimicrobials. It is the most Polymorphism (AFLP)
commonly used typing method. 3. Sequencing-based methods
4. Microarrays
Auxotyping: Based on nutritional requirement of the organism (e.g. Gonococcus)
Morphotyping: Based on different type of colonies in culture (e.g. Pseudomonas)
Serotyping: Based on the antigenic property of an organism.
• Streptococcus (Lancefield grouping)
• Based on capsular antigen, e.g. pneumococcus, meningococcus and influenzae
• Based on somatic antigen- E. coli, Shigella, Salmonella and Vibrio cholerae.

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Manila Adventist College
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