5.

Bacterial culture and identification

Competency

MI1.1: Describe the different causative agents of infectious diseases and the methods used in their
detection, and discuss the role of microbes in health and disease

Specific Learning Objectives: At the end the session, the students shall be able to,
 Explain the purpose of growing the bacteria in the laboratory
 Enlist the basic nutrients required in a culture medium for the growth of bacteria
 Enumerate various culture media used for growing bacteria
 Define different types of culture media (Enriched, Enrichment, Selective, Transport,
differential) and explain their uses
 Suggest the use of suitable culture media based on the type of clinical specimen and condition
 Enumerate the media, methods and uses of anaerobic culture
 Describe the media, methods and uses of blood culture
 Discuss the principle of automated blood culture systems with examples
 Compare and contrast the conventional from automated blood culture method
 Enumerate the methods/tests used for presumptive identification of bacteria from culture

Exercise 5:

1) Explain the purpose of growing the bacteria in the laboratory

The purpose of growing the bacteria in the laboratory is

a) Bacteria can be isolated in pure form from various infectious materials like blood, urine,
pus, sputum etc.
b) Culture helps to study the colonial characters and various other properties of bacteria.

2) Write the basic chemical constituents in a culture medium

The basic chemical constituents in a culture medium are

1. Nutrients -energy source- derived from oxidation and decomposition of protein and
carbohydrates.
Carbon source- ex: citrate
Nitrogen source- ex: peptone
2. Mineral salts like sulphates, phosphates, chlorides, carbonates of sodium, potassium,
magnesium and calcium.
3. A suitable pH usually between 7.2-7.6
4. Accessory growth factors.
Ex a) Tryptophan for S.typhi growth
b) X and V factors for the cultivation of Haemophilus influenzae.

3) Enumerate different types of transport media and indicate the clinical conditions and type
of bacteria that can be supported by such media
 Transport medium Clinical condition Bacteria

Aerobic:

1) Pike’s medium Streptococcal throat Streptococcus

infection

2) Amies medium, Gonorrhoea Neisseria

Stuart’s medium

3)VR (Venkatraman- Cholera Vibrio cholarae

Ramakrishnan medium)

Cary-Blair medium.

4)Buffered glycerol saline Shigellosis Shigella

Cary-Blair medium Salmonella.

Anaerobic:

1) VTM viral transport Covid-19 infection Corona virus

media

2) ATM anaerobic Dental anaerobic infection Fusobacterium, bacteroides

transport medium

4) Define different types of culture media (Enriched, Enrichment, Selective, differential) and
explain their uses

Type of media Examples Uses

Enriched Blood agar Used to test hemolytic property

media: of bacteria.

Chocolate agar Used to grow fastidious bacteria

like Haemophilus influenzae.

Loeffler’s serum slope Used for isolation of

Corynebacterium diphtheriae.

Enrichment Selenite F broth Shigella

media:
Tetrathionate broth Salmonella Typhi

Alkaline peptone water Vibrio cholerae

Gram negative broth Salmonella, Shigella

Selective Deoxycholate citrate agar For fecal samples

media:
Thiosulphate citrate bile sucrose For Vibrio species
 agar

Thayer -Martin medium To isolate N. gonorrhoeae

Lowenstein Jensen medium Mycobacterium species

Differential Mac Conkey medium To differentiate between lactose

media: fermenters and non-lactose
fermenters
A) Brain Heart Infusion broth (BHI broth)
B) Castaneda’s biphasic media
c) Bactec automated culture bottle.

6) What is blood culture? Enlist the media, methods and uses of Blood culture.
Recovery of microorganisms from blood in suspected cases of septicemia by using
appropriate blood culture media and blood culture methods is known as blood culture.

Various blood culture media available are:

1. Brain heart infusion broth (BHI) broth
2. Castaneda’s biphasic media
3. Bactec automated blood culture bottle.

Types of blood culture methods are:

A. Conventional blood culture methods, and
B. Automated blood culture methods.

Uses of blood culture are:

i. Blood culture is the culture of micro-organisms from blood for the laboratory
diagnosis of bacteremia.
ii. Infective endocarditis
iii. Useful in diagnosis of conditions associated with pyrexia of unknown origin.

7) Enumerate the automated blood culture systems. Add a note their principle
There are 3 automated blood culture systems
that are commercially available:

1. BacT/ALERT 3D
2. BacT/ALERT VITRUO
3. BACTEC.

Principle: It is based on fluorometric

detection of growth; uses an oxygen-sensitive
fluorescent dye present in the medium.
 8) Discuss the advantages and limitations of conventional and automated blood culture
systems

Blood culture system Advantages Limitations

Conventional Cheap There is higher risk of

contamination during
subculture.

Ability to observe colony Only blood sample can be

morphology in biphasic media. inoculated

Automated In addition to blood, CSF, High cost of instrument and

peritoneal, pleural and blood culture bottle.
synovial fluid can be
inoculated.

More sensitive, higher chances Inability to observe colony as

of recovery of liquid medium is used.
microorganisms.

9) List the anaerobic culture media

Robertson cooked meat media Tetrathionate broth Anaerobic gaspack

10) Describe the anaerobic culture methods and write their uses.
 Anaerobic culture methods include: Anaerobic culture is used to isolate anaerobes in :
 Production of vacuum dental infections
 By displacement and combustion of oxygen gas gangrene
 Absorption of oxygen by chemical methods- diabetic foot ulcer
 Anaerobic glove box infected bites
 By reducing agents
 PRAS (Pre-Reduced, Anaerobically Sterilized)


11) Give examples for presumptive identification of bacteria based on colony

characteristics.

Colony characters Probable bacteria

Golden yellow beta hemolytic colonies on blood agar Staphylococcus aureus

Green pigmented smooth colonies Pseudomonas aeruginosa

Alpha hemolytic colonies with green discoloration of Streptococcus species

surrounding medium on blood agar

Smooth greyish colonies showing swarming on blood agar Proteus species

12) Enumerate the biochemical tests used for presumptive identification of bacteria.

Catalase test Citrate test Methyl red test

Coagulase test Urease test Voges-Proskauer test

Oxidase test Triple sugar iron test Mannitol motility test

Indole test Sugar fermentation test Bile esculin hydrolysis test

Catalase test is used to differentiate between

Staphylococcus (catalase positive-effervescence is
produced) from Streptococcus (catalase negative).

It is positive for members of families

Enterobacteriaceae, Vibrionaceae, Pseudomonadaceae
Tube coagulase Slide coagulase
Due to coagulase enzyme Due to clumping factor
Done in tube Done in slide
Positive if clot is formed Positive if clumps are formed
Both tube and slide coagulase positive for S.aureus,
S.hyicus, and S.intermedius

Oxidase positive (deep purple)-

Examples include Pseudomonas, Vibrio,
Neisseria, Bacillus etc.
Oxidase negative (no colour change)-
Examples include; members of family
Enterobacteriaceae, Stenotrophomonas,
etc.

Indole positive- pink ring is formed.

Escherichia coli, Proteus vulgaris, Vibrio
cholerae etc.
Indole negative - Klebsiella pneumoniae,
Proteus mirabilis, Pseudomonas, Shigella,
Salmonella, etc.
 Urease test positive (pink colour) -
Klebsiella pneumoniae, Proteus
species, Helicobacter pylori, Brucella,
etc.
Urease negative - Escherichia coli,
Shigella, Salmonella, etc.

Citrate test is positive (blue colour)

for Klebsiella pneumoniae,
Citrobacter, Enterobacter etc.
Test is negative (green colour) for
Escherichia coli, Shigella,
Salmonella Typhi, etc.

Reactions in TSI Examples

Acidic slant/acidic butt ≥ 2 sugars fermented
Glucose fermented &
Lactose and/or sucrose fermented
A/A, gas produced, no Escherichia coli
H2S Klebsiella pneumoniae
Alkaline slant/acidic butt Only glucose fermenter group
K/A, no gas, no H2S Shigella
K/A, no gas, H2S S.Typhi
produced (small
amount),
K/A, no gas, H2S Proteus vulgaris
produced (abundant),
K/A, gas produced, H2S S.Paratyphi B
produced (abundant)
K/A, gas produced, no S.Paratyphi A
H2S
Alkaline slant/alkaline butt Non fermenters group
K/K, no gas, no H2S Pseudomonas
Acinetobacter
 Detects the ability of an organism to
ferment a specific carbohydrate
(sugar) incorporated in a medium
producing acid with/without gas.
Glucose, lactose, sucrose and
mannitol are widely used for sugar
fermentation.

In glucose phosphate broth, certain bacteria

ferment glucose to produce stronger acids that
maintain the pH below 4.4 which turns methyl red
indicator from yellow to red color
MR Positive (red color)-Escherichia coli
MR negative (yellow, i.e. no change in color)-
Klebsiella pneumoniae
In the presence of alkali and atmospheric oxygen,
acetoin is oxidised to diacetyl which reacts with α-
naphthol to give red colour
VP positive- Klebsiella pneumoniae, Enterobacter,
El Tor vibrios, Staphylococcus, etc.
VP negative - Escherichia coli, Shigella,
Salmonella, etc.

14) What is MALDI-TOF? Discuss its clinical utility in diagnostic bacteriology.

MALDI-TOF technology is automated system used for bacterial identification which

examines the pattern of ribosomal proteins present in the organism.
Use in diagnostic bacteriology:
i. Produce faster result.
ii. Can identify a wide range of organisms with accuracy