Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Cultivation
of Bacteria and Fungi
Applied Veterinary Bacteriology and Mycology:
Bacteriological techniques
Chapter 3:
Incubation Systems and Cultivation of Bacteria and Fungi
Author: Dr. J.A. Picard
Licensed under a Creative Commons Attribution license.
TABLE OF CONTENTS
INTRODUCTION .......................................................................................................................................... 2
SELECTION OF PRIMARY CULTURE MEDIA........................................................................................... 2
HANDLING OF SPECIMENS PRIOR TO INOCULATION .......................................................................... 2
INOCULATION OF MEDIA .......................................................................................................................... 3
INOCULATION ONTO SOLID MEDIA......................................................................................................... 3
INOCULATION INTO LIQUID MEDIA ......................................................................................................... 5
SWABS......................................................................................................................................................... 5
INOCULATION OF BIOPSY MATERIAL .................................................................................................... 5
Block technique ....................................................................................................................................... 5
Scraping Technique ................................................................................................................................. 5
For small specimens ................................................................................................................................ 6
URINE CULTURES ...................................................................................................................................... 6
INCUBATION ............................................................................................................................................... 6
INCUBATION SYSTEMS ............................................................................................................................. 8
REFERENCES: ............................................................................................................................................ 9
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Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Cultivation
of Bacteria and Fungi
INTRODUCTION
The type of primary media, atmospheric conditions and incubation temperatures are selected on the basis of
the nature of the specimen, case history and disease(s) suspected. Table 1 provides protocols for the
primary cultivation of bacteria from laboratory specimens (courtesy of Dr. M.M. Henton).
SELECTION OF PRIMARY CULTURE MEDIA
The most practical medium to use is blood agar, as it supports a wide variety of bacteria. Selective media are
used for the isolation of specific organisms or when severe bacterial contamination is suspected or known
e.g. a faecal specimen. Enrichment media are used when organisms in the sample are few, or have reduced
viability (sub-lethal).
It is often useful to inoculate the specimens, except for faecal specimens, into a tube of semi-solid brain-heart
infusion broth or Brucella broth, as this will support the growth of many fastidious organisms including
anaerobes in contaminated samples. However, there may be overgrowth of the pathogen(s) by other
bacteria.
HANDLING OF SPECIMENS PRIOR TO INOCULATION
1.
Should there be an unavoidable delay before processing of the specimens, store them at 4°C until
ready to commence. Some specimens can be frozen, e.g. milk for culture (not for somatic cell counts)
and lymph nodes for Brucella culture with little effect on the viability of the pathogens.
2.
Repeated thawing and freezing does however, reduce the number of viable organisms considerably.
Specimens intended for Mycoplasma isolation should not be frozen and neither should whole foetuses
as these can take some days to thaw out allowing putrefactive bacteria to grow in that period.
3.
Make notes of the overall appearance of the specimens, including details of the presence of excess
fluids, haemorrhages, pus, size abnormalities, specimen quality.
4.
Study the history carefully and take note of the suspected pathogens.
5.
If swabs arrive in a dried-out state in the laboratory, moistening with a little sterile water or saline may
assist recovery of the material.
6.
After the specimens have been processed it is important that they be stored in tightly closed containers
so as to reduce the risk of laboratory infections to personnel. Plastic disposable screw-topped bottles
(sputum jars) are well suited for this purpose.
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Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Cultivation
of Bacteria and Fungi
INOCULATION OF MEDIA
1.
When selective and non-selective media are being used for the same specimen, e.g. a swab, always
inoculate the non-selective medium first.
2.
Whenever possible it is sound practice to use solid media for primary isolations. Fluid media should
only be used if they are specifically designed to promote (enrich) the growth of a particular pathogen,
e.g. selenite brilliant green (SBG) broth for the isolation of Salmonella spp. The use of rich (nutritious)
non-selective fluid media is not recommended for primary isolation.
3.
Do not work in a draught which can lead to contamination and possible infection of workers or in direct
sunlight which can cause the destruction of bacteria
INOCULATION ONTO SOLID MEDIA
Material is streaked out using an inoculation loop (nichrome or platinum wire), swab or bent glass rod. The
inoculation loop should be sterilized in a flame until it glows red. The patterns most commonly used are
indicated in Figure 2 for primary cultures and Figure 3 for semi-quantitative cultures. The purpose of the
techniques shown is to dilute the inoculum sufficiently on the surface of the agar medium so that well isolated
colonies can be sub-cultured. Inoculation onto a selective medium requires a heavier inoculum. Inoculation of
an agar slant is performed by stabbing a straight wire into the medium to a depth of 2 - 3 mm from the
bottom, removing it and then streaking it on the surface in an S-motion, as in Figure 4. Semi-solid media are
inoculated with a stab action.
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Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Cultivation
of Bacteria and Fungi
Figure 2: Streaking of an agar plate
Figure 3: Streaking of an agar plate to obtain a matt of
growth
Figure 4: Streaking of an agar slant
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Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Cultivation
of Bacteria and Fungi
INOCULATION INTO LIQUID MEDIA
A broth tube is tipped at 30° and the inner surface of the glass is touched with the inoculation loop. When the
tube is returned to its upright position, the area of inoculation is submerged beneath the surface of the liquid.
SWABS
Make sure, when streaking the swab over the surface of an agar plate, that there is a sufficient inoculum on
the swab to obtain a satisfactory culture. Swabs can also be broken off into a fluid medium (e.g. into selenite
brilliant green for Salmonella).
INOCULATION OF BIOPSY MATERIAL
For large specimens, the “Block Technique” or the “Scraping technique” can be used:
Block technique
a)
Sear the surface of the organ lightly with a hot spatula.
b)
Cut out a small block in the center of the specimen (approx. 15mm x 15mm x 15mm) using a scalpel and
forceps that have been dipped in 70% alcohol and flamed in the Bunsen burner.
c)
Rub this block over about ⅓ of an agar plate and then streak out from the primary streak using an
inoculation loop so as to obtain single colonies after incubation
d)
A smaller block (approx. 3mm x 3mm x 3mm) may then be cut from the larger block and submerged in
enrichment broth, e.g. selenite brilliant green (SBG). Use about 1g tissue for 10ml SBG.
Scraping Technique
a)
Sear the surface of the organ lightly with a hot spatula.
b)
Make a 15mm deep incision into the organ (approx. 50mm in length, depending on the size of the
organ).
c)
Using a scalpel, scrape the sides and base of the incision until there is sufficient amount of tissue to use
as an inoculum. This inoculum can then be picked up using a swab or Pasteur pipette. For intestine, cut
through the intestinal wall and then scrape through the contents to expose the underlying mucosa. Now
scrape the surface of the mucosa using a cooled freshly flamed scalpel to obtain a sufficient amount of
tissue to form an inoculum. Use an inoculation loop to lift some of this tissue. Lightly streak the inoculum
over about one third of an agar plate. (Similarly, this inoculum may be used for fluid medium inoculation).
d)
Now use a flamed inoculation loop and streak out to obtain single colonies after incubation.
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Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Cultivation
of Bacteria and Fungi
For small specimens
The external surface is sterilized by dipping it into 70% ethanol and then passing it through a Bunsen burner
flame while holding it with a sterile forceps. It is then sectioned with a sterile scalpel blade and an impression
of the cut surface is made on the agar surface. This is spread further with an inoculating loop.
For very a small specimen it is best to mince or grind it with a sterile pestle in a mortar adding a small volume
of fluid media, such as brain-heart infusion broth. The whole specimen is used for inoculation onto the
selected medium.
URINE CULTURES
Before culture, examine the urine sediment for the presence of bacteria and inflammatory cells. Because of
the possible contamination of samples collected by catheterization or from mid-stream urine, bacteria in these
samples should be quantified. If the sample is collected by cystocentesis, it can be inoculated directly, without
the need for quantification.
INCUBATION
Incubate the cultures in the appropriate atmosphere (aerobic, anaerobic or microaerophilic) depending on the
pathogen suspected. Petri dishes are incubated in the inverted position, i.e. with the base facing upwards.
This prevents the formation of water droplets on the lid and from falling onto the solid medium. Incubate in a
humid atmosphere at 35 - 37°C, for at least 48 hours before reporting as negative when no growth has
occurred. Cultures should be examined after 18 hours or overnight incubation so that a preliminary result can
be given and sub-culturing be done before spreading bacteria such as Proteus or Bacillus spp. can overgrow
other bacterial colonies.
A 10 % CO2 atmosphere or candle jar may stimulate the growth of some aerobes such as Streptococcus
spp., Brucella spp., Corynebacterium spp., and some others. To ensure movement of CO 2 into the inoculated
Petri dishes, burn a 1-2mm groove into the edge of the base of the Petri dish using a flamed inoculation loop.
Low humidity incubation of solid media can lead to drying out. A relatively high humidity tends to stimulate
bacterial growth. Modern incubators are often fitted with humidifying devices, but if this facility is not fitted, a
small bowl of water may be placed in the incubator.
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Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Cultivation
of Bacteria and Fungi
Table 1: Cultural requirements of bacterial genera of veterinary importance
Family/Genus
Stain
Medium
Actinobacillus
Actinomyces
Bacillus anthracis
Bordetella
Brucella
Campylobacter
Chlamydophila
Clostridium
Corynebacterium
Dermatophilus
Enterobacteriaceae
Erysipelothrix
Fusobacterium
Haemophilus/Histophilus
Leptospira
Listeria
Moraxella
Mycobacterium
Mycoplasma
Nocardia
Pasteurella
Pseudomonas
Salmonella
Staphylococcus
Streptococcus
Treponema
Gram
Gram
Giemsa
Gram
mod ZN
Gram
mod ZN
Gram, FAT
Gram
Giemsa
Gram
Gram
Gram
Gram
Darkfield
Gram
Gram
ZN
Darkfield
mod ZN
Giemsa
Gram
Gram
Gram
Gram
FAT
Fragility of
organism
Cultural requirements
BA
BA
BA
BA, McC
S
S
S
BA
BA
BA
BA, McC
BA
BA/A
S
S
BA, S
BA/A
S
S
BA
BA, McC
BA
BA, McC
BA
BA
BA/A
Atmosphere
Aer
Anaer
Aer
Aer
CO2
CO2, H2
Note 3
Anaer
Aer
CO2
Aer
Aer
Anaer
CO2
Aer
CO2
Aer
Aer
CO2
Aer
Aer
Aer
Aer
Aer
Aer
Anaer
Time (days)
1
2-3
1
2
3-7
2-5
7
1-2
1-2
2-4
1
1
2-3
2-3
2w - 16w
2 - 3*
1-2
4w - 8w
7 - 14
3 - 15
1
1
1
1
1-2
3-7
Key:
ZN= Ziehl Neelsen, modZN= modified Ziehl Neelsen, FAT= Flourescence antibody test
BA = blood agar, BA/A= blood agar with antibiotics, McC= MacConkey agar, S= specialised medium
Aer= Aerobic, Anaer= anaerobic,
3. Tissue culture or embryonated hen’s eggs
- = Robust, no special precautions.
+ = May die unless care is taken
++= Delicate, special care should be taken in transit.
* Tissues may have to be held at 4°C for several weeks before culture can be carried out.
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+
++
++
+
+
+
++
+*
++
++
+
Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Cultivation
of Bacteria and Fungi
INCUBATION SYSTEMS
The types of specimens submitted to veterinary microbiology laboratories, and the procedure to be followed in
processing each depends on the disease or organism suspected. One of the problems often encountered in the
laboratory is to predict what pathogen or disease to suspect in the absence of a clinical history. Because information
is often lacking, routine procedures are generally established by the laboratory for the examination of the bulk of
specimens. If one does not know what bacteria to suspect and, as most bacterial pathogens are facultative
anaerobes, it is recommended that a CO2 incubator be used to provide 5-10% CO2 for the routine incubation of
plates containing non-selective media at 37°C. If such an incubator is not available, a candle jar will be satisfactory.
The CO2 content of a candle jar is usually between 3 – 5%.
The most important factors to be considered, excluding the kind of media to be used, are temperature, time and
the gaseous atmosphere in which the culture plates should be incubated. These will depend on the bacterial
pathogens that are being sought. The incubation temperatures, atmospheric conditions and times in Tables 2
and 3 are offered as general guidelines.
Table 2: Atmospheric and temperature requirements
Atmosphere
Normal atmosphere
(aerobic)
Incubation
temperature
Organism
37°C
Most of the pathogenic bacteria and the fungi causing a systemic mycosis. Strict aerobic
bacteria, such as Bacillus spp., Nocardia spp., and Pseudomonas spp., should preferably
be incubated under these conditions.
4°C
For cold enrichment of Listeria monocytogenes, Yersinia enterocolitica and Y.
pseudotuberculosis.
25°C
Primary isolation of most fungi, especially the dermatophytes.
28°C - 30°C:
Leptospira serovars
Borrelia spp.
42°C
Xylohypae bantiana (mould)
Salmonella enterica subspecies, Brachyspira species (to exclude contaminants).
Actinobacillus pleuropneumoniae
Actinomyces viscosus
Brucella spp. (optimally 10% CO2)
Dermatophilus congolensis (primary isolation)
5 - 10 per cent CO2
37°C
Francisella tularensis
Haemophilus spp.
Histophilus somni
Taylorella equigenitalis
Mycoplasma spp.
6% O2, 10% CO 2, 84%
N2
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25°C
Campylobacter fetus
37°C
Campylobacter fetus, C. jejuni and C. coli
Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques  Chapter 3: Incubation Systems and Cultivation
of Bacteria and Fungi
(microaerophilic)
42°C
Campylobacter jejuni and C. coli
Actinomyces bovis
Bacteroides spp.
Dichelobacter nodosus
Campylobacter mucosalis
37°C
Anaerobic
Clostridium spp.
Eubacterium species
Fusobacterium spp
Provetella melaninogenicus
Peptostreptococcus spp.
Brachyspira hyodysenteriae
42°C
Brachyspira hyodysenteriae
Table 3: Incubation times required for growth of bacteria
Time
Micro-organism
24 - 48 hours
Most of the rapidly growing bacteria.
48 - 72 hours
The rapidly growing bacteria when plated on selective media
Brucella spp.
Campylobacter spp.
4 - 6 days
Nocardia asteroides
Mycoplasma spp.
The relatively fast growing fungi
2 - 3 weeks
3 - 8 weeks
4 - 16 weeks
Most of the dermatophytes (T. verrucosum up to 5 weeks)
Mycobacterium avium
Mycobacterium bovis
Blastomyces dermatitidis and Histoplasma capsulatum
Mycobacterium paratuberculosis
REFERENCES:
1. Quinn, P.J., Markey, B.K., Leonard, F.C., FitzPatrick, E.S., Fanning, S., Hartigan, P.J. Veterinary
Microbiology and Microbial Disease, (2011). Wiley-Blackwell. ISBN 978-1-4051-5823-7
2. Quinn, P.J., Carter, M.E.; Markey, B., Carter, G.R. Clinical Veterinary Microbiology. 1994. Wolfe. ISBN 0
7234 1711 3.
3. Carter, G.R. and Cole, J.R. Diagnostic Procedures in Veterinary Bacteriology and Mycology. Fifth Edition.
1990. AcademicPress.ISBN0-12-161775-0.
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