Professional Documents
Culture Documents
[HIGHER]
Margot McKerrell
abc
Acknowledgement
Learning and Teaching Scotland gratefully acknowledge this contribution to the National
Qualifications support programme for Biotechnology. The advice of Jim Stafford is
acknowledged with thanks.
First published 2004
Learning and Teaching Scotland 2004
This publication may be reproduced in whole or in part for educational purposes by
educational establishments in Scotland provided that no profit accrues at any stage.
ISBN 1 84399 058 X
CONTENTS
Introduction
Section 1:
Section 2:
Culturing techniques
15
Section 3:
Identification of micro-organisms
37
Bibliography
41
Appendix:
45
INTRODUCTION
This unit introduces you to some of the techniques that are used to
study micro-organisms. A micro-organism is any small organism that
cannot be clearly seen without the help of a microscope. The microorganisms that you will use to carry out the study of microbiological
techniques include bacteria, fungi and viruses. Many biotechnology
processes rely on the use of micro-organisms and so it is important that
you know how to work safely with them. That is why this unit is included
in Higher Biotechnology.
These student notes will provide you with the knowledge and
understanding that you will need to carry out microbiological
techniques.
SECTION 1
correct temperature or time is not observed, then the items inside the
autoclave cannot be guaranteed to be sterile. To ensure that the correct
autoclaving procedure has been carried out, autoclave monitors such as
Brownes tubes and test strips, are added to the autoclave with the items
to be sterilised and then checked at the end of the procedure. In
general, these monitors change colour to show that sterilisation has
taken place; for example, Brownes tubes change from red to black
when they have been sterilised correctly.
The autoclave is used to sterilise objects and materials that are heatresistant such as glassware, cloth, rubber, metallic instruments, liquids,
paper and heat-resistant plastic. In the laboratory in your school or
college, you may use an autoclave to sterilise culture media, scalpels and
glassware such as test tubes and conical flasks.
Another technique that is used to sterilise materials and objects is the
use of dry heat. Dry heat sterilisation takes place in a dry oven that has
electrical coils that radiate heat. Dry heat kills micro-organisms by
dehydrating them and denaturing their proteins. Higher temperatures
are needed when using dry heat compared to using steam. In general,
items are sterilised at 160C for two hours.
Dry heat is used to sterilise items that may be adversely affected by
steam, such as powders, oils, glass pipettes and metal instruments that
may corrode if exposed to moisture. Dry heat is not suitable for plastic,
cotton, paper or for solutions that would boil and dry out, such as
culture media.
Some liquids cannot be sterilised by autoclaving or in dry-heat ovens
because the temperatures used in these techniques cause the
components in the liquids to denature. Heat-sensitive liquids can be
sterilised using filtration.
One type of filtration uses a filter that is composed of a mass of fibres.
When liquid is passed through this type of filter, any micro-organisms
present in the liquid adsorb (stick) to the fibres. Another type of
filtration uses a filter that has pores (holes) large enough for liquid to
pass through but too small to let micro-organisms through, so they
become trapped on the filter. These filters are known as membrane
filters.
Filtration is used to sterilise solutions containing antibiotics, enzymes
and glucose. It is also used to sterilise air, for example before it is
pumped into a fermenter.
10
Multi-oxidising detergents
These destroy a wide variety of micro-organisms.
They work by denaturing proteins.
They are used in the laboratory for a variety of purposes such as
swabbing benches and in disposal containers for used glass pipettes.
11
12
You have now finished the first part of this unit and you should be
familiar with the procedures used to prevent the unwanted growth of
micro-organisms. You should be familiar with methods of sterilisation
and disinfection; know the difference between a hazard and a risk; be
aware of control procedures that you can carry out to minimise the
risks of working with micro-organisms; know how to deal with smallscale spillages; and how large-scale spillages of broth are contained.
You are now ready to be introduced to some of the techniques that are
used to grow micro-organisms.
13
14
CULTURING TECHNIQUES
SECTION 2
Culturing techniques
This section of the unit introduces you to the theory behind the
techniques that are used to culture micro-organisms. However, you
should remember that this unit is a hands-on practical unit and you will
be given the opportunity to carry out many of the techniques in the
laboratory so that you will become competent in the safe handling of
micro-organisms. Your tutor will provide you with the protocols and
methods that you need to carry out the techniques safely.
While working with living micro-organisms you must develop and use
good working/laboratory practices. These practices are important for
several reasons:
to ensure that you do not contaminate yourself or others in the
laboratory, or contaminate the laboratory itself
to avoid contaminating the cultures with which you are working
to prevent accidentally taking micro-organisms out of the laboratory.
Aseptic technique is the name given to all the procedures that are used
when working with micro-organisms to prevent contamination. Some of
the procedures that are used and the reasons for them are described
below.
Firstly, it is essential that you prepare yourself in readiness for carrying
out practical work with micro-organisms. Long hair must be tied back,
hands must be washed with soap and water, cuts covered with
waterproof plasters (alternatively plastic gloves can be used) and
personal protective equipment such as a lab coat and eye protection
must be worn. You must ensure that the sleeves of your lab coat are
rolled down and that it is buttoned up to protect your normal clothes
from accidental spillages.
Next, you must prepare your work space. Work benches used for
microbiology must be smooth and non-absorbent so that they do not
become contaminated if there is a spillage. If necessary, benches can be
made suitable for microbiology work by covering them with nonabsorbent material such as benchcote. Before starting work, bench
surfaces are always disinfected to reduce the possibility of contaminating
the cultures with which you are working. It is good practice to have a
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CULTURING TECHNIQUES
16
CULTURING TECHNIQUES
17
CULTURING TECHNIQUES
Nutrient
Nitrogen
To make proteins
and nucleic acids
Carbon
To make carbohydrate,
proteins, nucleic
acids and lipids
As a source of energy
Phosphorus
Phosphate ions
Sulphur
To make proteins
Sulphate ions
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CULTURING TECHNIQUES
Complex media are those in which the nutrients obtained from the
ingredients are not present in defined (known) quantities. For example,
some complex media contain peptone, which is a mix of proteins that
have been partially hydrolysed (broken down). Peptone is a source of
carbon, nitrogen and sulphur but the actual quantities may vary from
batch to batch. Other complex media contain yeast or beef extract,
which are sources of amino acids, sugars and vitamins.
Synthetic media are those in which pure chemical components are
added in known quantities. An example of a synthetic medium is Czapek
Dox that is used to culture fungi. The quantity of ingredients used to
make a volume of 1000cm3 is shown below:
Sodium nitrate
Potassium chloride
Magnesium glycerophosphate
Iron sulphate
Sucrose
Agar
20.0g
0.5g
0.5g
0.01g
0.35g
12.0g
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CULTURING TECHNIQUES
Sub-culturing
technique
Culture medium
that micro-organisms
are taken from
Solid to solid
Agar plate
or
Agar slope
Agar plate
or
Agar slope
Solid to liquid
Agar plate
or
Agar slope
Liquid broth
Liquid broth
Agar plate
or
Agar slope
Liquid broth
Liquid broth
Liquid to solid
Liquid to liquid
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CULTURING TECHNIQUES
21
CULTURING TECHNIQUES
22
CULTURING TECHNIQUES
Pure cultures
In laboratories, pure cultures are normally used and there are many
reasons for this:
When studying the characteristics of a micro-organism, other microorganisms should not be present. Otherwise you will not know for
certain that it is the micro-organism being studied that is carrying out
the reaction.
Contaminating micro-organisms use up nutrients in the medium, so
there is less nutrient and so less growth of the actual micro-organism
being studied.
Contaminating micro-organisms may produce substances that prevent
the growth of the micro-organism being studied.
Pure cultures are also used in industrial fermentation where a
commercial product is being made. Contamination in a fermentation
process may lead to reduced product being formed (if there is less
growth of the micro-organism carrying out the fermentation), or the
product may be impure because of the presence of substances produced
by the contaminating micro-organism.
There are a number of ways that pure cultures can be obtained from a
mixed culture, for example by using selective and differential media and
by exploiting discrete growth characteristics of the micro-organisms.
A selective medium contains one or more ingredients that prevent the
growth of certain micro-organisms but not others. These media are
important in the isolation of one type of micro-organism from samples
containing lots of types of micro-organisms such as those found in
faeces, saliva, water and soil.
Mannitol salt agar is a selective medium that contains high
concentrations of salt. This prevents the growth of most human
pathogens (bacteria that cause disease) but the bacterium
Staphylococcus grows in this medium, so it is used to select for this
micro-organism.
MacConkey agar is a selective medium that contains bile salts. This
medium can be used to select for bacteria (such as E. coli) that normally
grow in the human intestines as they can grow in the presence of bile
salts.
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CULTURING TECHNIQUES
Enumerating micro-organisms
Enumerating micro-organisms means counting the number of microorganisms in a given sample. Counting micro-organisms is not always
easy because of the large number of micro-organisms that can be
present, even in a relatively small sample. For example, a small drop of
liquid culture of bacteria could contain as many as several million
bacterial cells. It is impossible to give the exact number of microorganisms that are present in a sample, so an estimate of the number of
micro-organisms is given.
A sample with many micro-organisms is diluted so that a smaller number
of micro-organisms is present, which is easier to count. The number of
micro-organisms in the diluted sample is then multiplied by the dilution
factor to give an estimate of the number of micro-organisms in the
original, undiluted sample. For example, if a sample is diluted by 105 (1
in 100,000) and the number of micro-organisms in the diluted sample is
25, then the original sample has 25 100,000 = 2.5 106 microorganisms in it.
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CULTURING TECHNIQUES
25
CULTURING TECHNIQUES
Direct methods of enumeration involve counting the number of microorganisms directly, for example by counting the number of colonies on
an agar plate or by using a microscope to count the number of cells
observed.
Indirect counts can be made by growing micro-organisms in liquid
broth. As micro-organisms grow, the broth becomes cloudy or turbid.
The turbidity is measured using a device such as a colorimeter or a
spectrophotometer.
Direct counting of micro-organisms using a haemocytometer
A haemocytometer is a counting chamber as shown in Figure 4.
Figure 4
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CULTURING TECHNIQUES
From this diagram, you can see that micro-organisms sometimes lie
between one large square and the next. It is standard practice to count
micro-organisms on the top and right-hand side of the square, but to
ignore the bottom and left-hand side.
From Figure 5:
5 micro-organisms are counted in one large square,
so there are 5 micro-organisms in 0.004mm3 sample
5 1
= 1250 micro-organisms per mm3
0.004
27
CULTURING TECHNIQUES
If the original sample was diluted, the dilution factor must be taken into
account, for example if the original sample was diluted 1 in 1000 (103),
then the original sample would contain 1.25 10 6 1000 = 1.25 109
micro-organisms per cm 3.
Indirect counting of micro-organisms using a colorimeter or
spectrophotometer
The total number of micro-organisms can also be estimated by indirect
counting using a colorimeter or spectrophotometer. The number of
micro-organisms in a suspension can be estimated from a turbidity
reading on a colorimeter or spectrophotometer. These instruments
measure the cloudiness (turbidity) of a suspension of micro-organisms.
The more micro-organisms there are in the suspension, the higher the
turbidity, so the higher the optical density reading from the
instruments.
The total number of micro-organisms in a suspension can be worked out
from the turbidity reading if a standard curve is available. A standard
curve is generated by obtaining the optical density of micro-organisms of
known number in a colorimeter or spectrophotometer. This data is
plotted to form a standard curve. An example of a standard curve is
shown in the graph on the next page (Figure 6).
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CULTURING TECHNIQUES
Figure 6
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CULTURING TECHNIQUES
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CULTURING TECHNIQUES
31
CULTURING TECHNIQUES
This figure shows that this bacteria is sensitive to penicillin but resistant
to tetracycline.
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CULTURING TECHNIQUES
Tissue culture
It is possible to culture isolated plant tissue in the laboratory using
techniques similar to those for culturing micro-organisms. When
culturing plant tissues, strict aseptic technique is needed to maintain
sterile conditions and to prevent contamination of the plant cells by
micro-organisms.
Plant tissue culture can be used to produce thousands of identical
plants. It allows commercial plant growers to produce clones of plants
such as pineapple, rose, orchid and palm oil in a relatively short period
of time. Another advantage of producing plants in this way is that they
are pathogen-free (this means that they are not diseased).
One method by which plants are micropropagated is by isolating a piece
of plant tissue, called an explant, and culturing it under sterile
conditions, either in agar or liquid medium. If the explant is given the
correct nutrients, vitamins and plant growth substances, the cells of the
explant first divide into a mass of undifferentiated cells, called the
callus, then the cells differentiate and finally they develop into a plant.
Plants can also be micropropagated from the apical meristem of a plant.
The apical meristem is the tissue found at the tip of the shoots. The cells
in this tissue are actively dividing and produce new growth of stems.
These cells are normally free from pathogenic (disease causing) microorganisms, so plants regenerated from them are pathogen-free.
The meristem is removed from the plant and sterilised with disinfectant.
It is then placed on agar containing nutrients until a shoot develops.
The shoot is placed in agar containing plant growth substances that
induce the development of roots. When roots have developed, the
plantlets are planted in sterile compost.
The nutrient medium used in micropropagation contains a variety of
substances essential for growth. Some of the substances found in the
nutrient medium are described below:
It contains a source of carbon. A callus does not have chlorophyll,
the pigment which is involved in photosynthesis (the process that
produces sugar in a plant). Thus, the nutrient medium contains a
source of carbon, usually in the form of a sugar. The sugar is used by
the callus in aerobic respiration, the process that provides the plant
with energy in the form of ATP.
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CULTURING TECHNIQUES
(b)
(c)
34
CULTURING TECHNIQUES
(d)
(e)
35
36
IDENTIFICATION OF MICRO-ORGANISMS
SECTION 3
Identification of micro-organisms
One of the most fundamental tasks that someone working with microorganisms must perform is to identify micro-organisms. There are a
number of ways that this can be done. Firstly, the micro-organism can be
stained and viewed under the microscope. This gives information
regarding the shape (morphology) of the micro-organism. Another way
of identifying micro-organisms is to find out the reactions that the
micro-organism carries out. This is known as biochemical testing.
Once the morphology and biochemistry of the micro-organism is
known, identification keys and tables can be consulted to work out the
identity of the micro-organism being investigated.
Use of the microscope in identifying micro-organisms
Micro-organisms are often difficult to observe using a microscope and so
they are generally stained before being viewed.
A stain that is commonly used in the initial identification of a bacterium
is the Gram stain. It identifies bacteria as being gram positive or gram
negative, depending on the type of cell wall present in the bacterium.
Gram-positive bacteria appear purple when viewed under the
microscope whereas gram-negative bacteria appear red.
(The gram stain is also discussed in Section 1 of the Student Materials
for Unit 1: Microbiology see page 8.)
When viewed under a microscope, bacteria are observed to have a
definite shape (morphology). Three shapes are commonly seen
round, rods and spirals.
Figure 11
Round bacteria are called cocci:
37
IDENTIFICATION OF MICRO-ORGANISMS
Biochemical tests
These tests give information about the reactions that a micro-organism
carries out, such as whether it requires oxygen for growth or whether it
ferments a particular sugar to produce acid. Each species of bacterium
has a characteristic profile or fingerprint of biochemical tests that can
be used to identify it.
Some of the tests that are carried out are described in the following
paragraphs.
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IDENTIFICATION OF MICRO-ORGANISMS
Extracellular digestion
In these tests, micro-organisms are inoculated on different types of
medium. If the micro-organism produces extracellular enzymes, then it
is able to grow on the medium. Examples include growing microorganisms on media containing starch, casein, gelatine and fat.
For example, if a micro-organism is inoculated onto agar medium
containing starch and if it produces amylase (which digests starch), then
the micro-organism grows on this medium. The digestion of the starch
can be observed by staining the agar plate with iodine.
Fermentation of carbohydrate
Micro-organisms are grown in media containing different carbohydrates
and the production of acid or acid and gas is recorded. For example,
the following table shows the characteristic results of two different
bacteria when grown in the presence of glucose and lactose:
Bacteria
Lactose fermentation
Acid
Gas
produced
produced
Glucose fermentation
Acid
Gas
produced
produced
E.coli
Shigella
Catalase test
A sample of viable bacteria is mixed with a solution of hydrogen
peroxide. If catalase is present, oxygen is produced and frothing of the
sample is observed.
39
IDENTIFICATION OF MICRO-ORGANISMS
Oxidase test
Cytochrome c is used to test for the presence of the enzyme oxidase.
Flowcharts are used to identify bacteria based on their morphology and
biochemical tests. An example of a flow chart is shown:
Figure 12
From this chart, a bacterium that is gram positive, catalase negative and
found in a chain arrangement can be identified as being Streptococcus.
40
BIBLIOGRAPHY
41
BIBLIOGRAPHY
42
BIBLIOGRAPHY
43
44
APPENDIX
These support materials are available for students and there are also
separate materials for lecturers/technicians.
HSDU/SSERC Biology/Biotechnology Microbiology Techniques
(Intermediate 1 Advanced Higher) support materials is a step-by-step
manual that provides a number of detailed protocols for techniques
required in this unit, such as:
Aseptic technique
Media preparation
Subculturing
Staining
Enumerating micro-organisms.
45