WEEK 04

MICROBIAL CULTURING METHOD

OBJECTIVE
To understand the importance of correct culturing methods in microbiology

LEARNING OUTCOMES
1. To explain the principles behind the use of solid and liquid growth media for
microbial cultivation
2. To perform correct culturing method using appropriate tools and techniques
3. To identify and describe different colony morphologies
4. To evaluate the purity of a culture
5. To collect, tabulate, analyse and evaluate the experimental data
6. To collaborate with companion students in both experiments as well as in
documentation of the experiment

INTRODUCTION
The general requirement for all microbiologists is the reproducible growth of their
microbial cultures as well as being free from contaminating chemicals or other
microorganisms. Culture media provide the nutrient requirements whilst the
aseptic techniques maintain sterility and reduce the possibility of contamination
during microbiology experiments. Since microorganisms are naturally ubiquitous
(everywhere in our surroundings) – in the air, soil and water, the aseptic
technique is an indispensable technique that all microbiologists must master.

To grow a microorganism in any given growth medium, microbial inoculation is

performed. Inoculating microorganisms from one medium to another (or from a
food sample into a growth medium) is usually done using a metal wire (inoculating
needle) or loop (inoculating loop) as shown in Figure 1 a. Platinum or nichrome
wires of different gauges are used. Nichrome has oxidising properties and hence
in some of the tests where this property of bacterium is to be tested (e.g. oxidase
test), platinum wire, instead of nichrome should be used. This wire is sterilised by
holding it vertically in the flame of the burner so that the whole length of wire
becomes red hot (Figure 2). It is then allowed to cool down before it touches any
material suspected to be having bacteria to avoid the heat killing the organisms.

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Pre-sterilised disposable loops (Figure 1 b) are now available commercially. The
wire can be used as a:

 Straight wire to stab the culture, picking of single colonies as well as for
inoculating the liquid media
 Thick wire which is useful for lifting viscous material such as sputum
 Wire loop which is usually of 2 mm diameter is most useful of all inoculating
wires. This is preferred to seed a plate of medium as the straight wire usually
cuts the agar.

Figure 1: (a) metal (reusable) inoculating loop and needle, (b) plastic
(disposable) inoculating loop and needle.

Figure 2: Sterilisation of inoculating loop is done using the blue flame of a

Bunsen burner until the loop is flaming red.

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The mouth and neck of a culture bottle must also be flamed after the removal of
its cap and before recapping to kill any microorganisms on the rim of the bottle.
These steps are among the basics in performing aseptic techniques.

TYPES OF CULTURES

a) Broth culture
The microorganism grows in a liquid medium

b) Slope / slant culture

The microorganism grows in a test-tube or small bottle where agar medium
was solidified at a slant. The bottle screwcap should be loosened during
incubation to allow for oxygen. Slope cultures are usually used to maintain
cultures for long term storage.

c) Stab culture
The microorganism grows in a tube or bottle in solid medium. Inoculum is
stabbed into the middle of the solid medium using a straight wire. Stab
cultures are used in some characterisation exercises to study lateral
outgrowth (motility). Also known as motility agar.

d) Plate culture
The microorganism grows on the surface of or inside of the solid medium in
a Petri dish. The term “plating” refers to the inoculation technique on the
surface of agar in a Petri dish.

Figure 3 illustrates the various types of culture media.

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Figure 3: Liquid medium or broth (a); slope / slant medium (b); stab medium
(c); and agar plate (d).

TYPES OF PLATING TECHNIQUES

a) Streak-plating
The inoculating loop or wire is used to transfer the microorganism as a
straight line on the surface of the agar.

b) Dilution streak technique

The dilution streak technique is used to obtain separated colonies of
microorganisms with the objectives of producing pure microbial cultures or to
study colony morphology.

c) Lawn-plating / spread-plating
The microorganism from broth culture or liquid sample is spread onto the
surface of solid media with an L-shaped sterile glass spreader (also known
as bent hockey stick).

d) Pour-plating
The microorganism is cultured on the surface and inside of the agar. The
liquid inoculum is pipetted onto a sterile Petri dish. Next, 15 – 20 mL of
molten agar is poured onto the Petri dish and gently swirled to mix the
culture into the medium. The agar is then allowed to set/solidify and then the
culture is incubated. This method is usually used to conduct viable counts.

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PLATING PROCEDURES

1. Dilution streak plating

The aim of streak plating is to distribute or spread the inoculum onto the surface
of the medium to separate the bacterial cells from each other. The resulting
bacterial growth should then occur in colonies which are separated from each
other.
i. Be sure to only slightly lift the lid of the petri dish and the opened side facing
away from you (to reduce contamination from the air, be sure to replace the
lid as soon as you have finished streaking).
ii. Take a small amount of inoculum with a sterile wire loop and spread onto
area A (Figure 4).
iii. Sterilise you wire loop and then make two to three parallel lines away from A
towards a fresh area of the agar (B). Make the parallel lines about 5 mm
from each other.
iv. Repeat the streaking several more times to areas C, D and E as shown in
Figure 4. Remember to sterilise the wire loop between each sequence. At
each step, the inoculum is derived from the most distal part of the
immediately preceding strokes so as to gradually reduce the number of
bacteria. This helps in obtaining isolated colonies.

Figure 4: Seeding / inoculating a culture plate by the dilution streak plating

technique.

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 2. Slope culture

Using aseptic techniques the slopes of solid media are inoculated by streaking
the surface of the agar with loop in a zig zag manner beginning at the bottom of
the slope and pulling the wire loop up.

3. Culture broth

i. Loosen the test tube cap, if necessary, but do not remove it.
ii. Hold the loop in your dominant hand and flame-sterilise it. Allow the loop to
cool a few seconds. (Figure 5a)
iii. Pick up the test tube in your non-dominant hand. Use the little finger of the
hand holding the loop to remove the cap from the test tube. Do not set the
cap down. (Figure 5b)
iv. Flame the mouth of the tube (Figure 5c).
v. Insert the loop into the tube and pick up a loopful of culture (Figure 5d).
vi. Remove the loop, again flame the mouth of the tube and carefully replace
the cap on the tube.
vii. Return the tube to the rack.
viii. Pick up the tube you wish to inoculate.
ix. Remove the lid as before, again holding the lid in the little finger of the hand
holding the loop.
x. Flame the mouth of the tube.
xi. Slide the loop into the tube.
xii. Remove the loop and carefully replace the cap on the tube after flaming the
mouth of the tube again.
xiii. Return the tube to the rack.
xiv. Flame the loop and set it down.

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 Figure 5a Figure 5b

Figure 5c Figure 5d

Important points on inoculation of culture media

 Aseptic technique is important to avoid contamination.
 When more than one medium is inoculated, follow a particular order.
Inoculate media without inhibitors, followed by indicator and then selective
media.
 While processing fluid specimen, inoculate liquid media first to reduce the
chances of carryover from contaminated solid media.
 Properly label the media to be inoculated to avoid any mix-up of the
specimens.
 Inoculate the media with clinical specimens as soon as possible.

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 Minimise the aerosol production by opening the caps of liquid media slowly,
avoiding vigorous shaking of the specimen and avoiding the expulsion of the
last drop from the pipette.

Microorganisms may show distinguishing morphologies when cultured on different

growth media. This macroscopic appearance (macromorphology) is often used for
their preliminary identification. Agar slopes / slants, agar plates, deep gelatine,
and nutrient broth are commonly used for this.

MATERIALS AND METHODS

MATERIALS
Bacterial cultures : Bacillus cereus in broth medium
: Staphylococcus aureus on agar medium
Deep nutrient agar
Nutrient agar
Nutrient broth
Nutrient gelatine
Nutrient slant

APPARATUS
Inoculating loop
Inoculating needle

PROCEDURES
1. On separate Nutrient agar plates, perform streaking using the given bacterial
cultures.
2. On separate Nutrient agar plates, perform dilution streaking using the given
bacterial cultures.
3. On separate Nutrient slants, perform streaking in a straight line using the
given bacterial cultures.
4. In separate tubes of Nutrient broth, transfer a loopful of bacterial culture.
5. For deep nutrient agar, stab a single line with inoculating needle through the
surface into the middle of the agar. Carefully pull out the needle out the way
it went in.
6. For nutrient gelatine, stab a single line with inoculating needle through the
surface into the middle of the agar. Carefully pull out needle out the way it
went in.

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7. Incubate all inoculated plates at 37C for 24 hours. Following the incubation
period, observe and record all observations except for nutrient gelatine
which should be re-incubated at 20C for another hour. Observe the
liquefaction / solidification of the nutrient gelatine.

RESULTS
Observe and take notes of the growth patterns of the cultures in the various
growth media. Use the following notes and charts as your guide.

OBSERVATION CHART

1. Nutrient slant

i) Observe the amount of growth –

none = 0, slight = +, moderate = ++, abundant = +++.

ii) Coloration - Two types of pigmentation may occur:

a) pigmentation occurring within the organism itself; or
b) water soluble pigment that diffuses into the surrounding medium.

Most organisms will lack chromogenesis (pigment production), exhibiting a

white, beige, or grey growth. Pigmentation within the organism may be red,
yellow, violet, or other colours. Soluble pigments may be blue, green,
yellow, brown, or other colours. Hold the slant up to the light to examine for
diffusible pigments. It may be helpful to compare the colour of the agar with
an uninoculated slant.

iii) Opacity - Surface growth can be termed as opaque, as transparent, or as

translucent (partial transparency) depending on the degree of growth.

iv) The macroscopic appearance of the growth from the single streak
inoculation is described by comparing it to the drawings shown in Figure 6.
a. filiform - uniform growth along the line of inoculation.
b. echinulate - margins of growth have a toothed appearance.
c. beaded - separate or semi-confluent colonies.
d. effuse - growth is thin, veil-like, unusually spreading.
e. arborescent - branched, tree-like growth.
f. rhizoid - root-like appearance.

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 Figure 6: Microbial growth patterns on slant / slope agar.

2. Nutrient broth

Growth patterns in broth may be used to distinguish between microorganisms

during identification. These patterns can be characteristic for certain species.
Remember, however, that these growth patterns can be influenced by growth
conditions such as type of medium used or the temperature of incubation.
Additionally, the growth may be disturbed accidentally by shaking or moving the
tube.

i. Surface
A pellicle type of surface differs from the membranous type in that the latter
is much thinner. A flocculent surface is made up of floating adherent masses
of bacteria. (Figure 7)

ii. Subsurface
Below the surface of the broth, the medium may be described as one of the
following:
a. turbid - the medium is cloudy.
b. granular - the medium has small particles visible in suspension.
c. flocculent - the medium has small masses visible in it.
d. flaky - the medium has large particles visible in suspension

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 Figure 7: Surface growth patterns in nutrient broth.

3. Deep nutrient agar

This test is used to demonstrate the ability of bacteria to move and is the
presumptive test for the presence of flagella. To be considered motile, the growth
of the bacteria must extend outward (Figure 8) from the line of inoculation in all
directions. It may be helpful to compare your inoculated tube with an uninoculated
one.

Figure 8: Growth patterns along stab line in deep agar.

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4. Nutrient gelatine

This test is used to determine the ability of a microorganism to produce hydrolytic

exoenzymes called gelatinases that digest and liquefy gelatine. The presence of
these enzymes, as determined by the liquefaction, is used for identifying certain
bacteria. The exoenzyme gelatinase will hydrolyse gelatine, a protein derived
from collagen. The enzymes first hydrolyse the gelatine into polypeptides, and
then further break down the polypeptides into the smaller amino acid molecules.
These can then be easily transported into the bacterial cells. See Figure 9.

Once the tubes are inoculated, they will be incubated. This will cause the gelatine
to melt. In order to determine whether or not the reaction has taken place, the
incubated tubes must first be refrigerated until the control tube is once again solid.

Figure 9: Patterns of gelatine hydrolysis.

5. Nutrient agar
Colonies on NA plates should be studied with respect to their colour and opacity,
and for their configuration, elevation, and margin (Figure 10). Do not open the
plates during observation.

Identifying and categorising different isolated bacterial colonies based upon

varied appearance and morphology (form and structure) will permit the selection
and transfer of different species from a mixed culture, and allow transfer of a
single colony to a sterile medium for cultivation of a pure culture. This exercise

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also shows how many diverse bacteria and fungi are present in our environment,
as exhibited by the varied morphologies.

When a single bacterial cell is deposited on the surface of a nutritive medium, it

begins to divide exponentially. After thousands (up to billions) of cells are formed,
a visible mass appears. This mass of cells is called a colony. Each species of
bacteria or fungi will exhibit characteristic colonies.

Colony characteristics are described in standard terminology to permit

interpretation by other microbiologist. These terms may appear to be complicated;
however, you will soon become familiar with them. Remember: the best
descriptions are complete, but concise.

Common terminologies:
1. Colony shape and size : round, irregular, punctiform (tiny)
2. Margin (edge) : entire (smooth), undulate (wavy), lobate (lobed)
3. Elevation : convex, umbonate, flat, raised
4. Colour : colour or pigment, plus opaque, translucent, shiny or
dull
5. Texture : moist, mucoid, dry (or rough).

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Figure 10: Colony appearances on agar plate.

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Table 1: Observation of different culturing methods on different growth media.

Growth medium / Bacillus cereus Staphylococcus aureus

culturing method (liquid source) (solid source)

Motility agar / stab agar /

deep agar
(for stabbing)

Nutrient agar
(for streaking)

no need, as the inoculum

Nutrient agar
for spreading must come
(for spreading)
from liquid source

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 no need, as the inoculum
Nutrient agar
for pour-plating must
(for pour-plating)
come from liquid source

Nutrient broth

Nutrient gelatine

Nutrient slant

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DISCUSSION

1. Explain why we culture microorganisms.

2. Explain the purpose of slope, stab, streak-plate, spread-plate, and pour-plate
culture techniques.
3. Explain what is aseptic technique, and why it is crucial.
4. Explain the significance / impact of Bacillus cereus in the food industry.
5. Explain the significance / impact of Staphylococcus aureus in the food
industry.

QUESTIONS

1. What is the difference between liquid and solid growth media?

2. What would happen if you did not flame your loop between different the
plate?
3. Why do you pass the mouth of the tube through the flame?
4. The absence of living microorganism is called:
a. Sepsis
b. Sterility
c. Contamination
d. Pathogenic
5. When microorganisms are present on tissue or a surface, it is called:
a. Contamination
b. Cross-contamination
c. Partial Contamination
d. Asepsis

REFERENCES
1. List all the references you cite in preparing the report.
2. Make sure you used recent references (year 2000 onwards).
3. Preferably use journal articles as references. Although accepted, keep
online references to a minimal.

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