PRACTICAL 1-BACTERIAL STAINING AND COLONY CHARACTERISTICS INTRODUCTION Just as different plants and animals have various morphologies or shapes, so do bacteria, both microscopically and microscopically. The observing of bacteria colonies or the examination of plate cultures are very important since bacteria were majorly first cultured on solid media such as agar media. In addition, this technique is very important since different bacteria possesses different morphology and might be used to differentiate between microorganisms in further test. In this practical, 13 types of bacteria cultures were given and their morphology was observed such as size, margin, elevation, colour, appearance and opacity. The further step in order to examine the bacterial morphology is the staining technique. A stain or dye is a molecule that can bind to a cellular structure and give it colour. Staining techniques are very important as it can be used to investigate the structures, the chemical reaction towards the cell and thus, categorise it. There are two types of dyes, the cationic or basic dyes and anionic dyes. The examples of cationic dyes are crystal violet and malachite green and the anionic dyes are eosin and picric acid. The staining techniques consist of three types; simple staining (used only one type of dye) and differential staining (used more than one dye) and special staining. The examples for differential staining are Gram stain, Ziehl-Neelsen acid-fast stain and negative stain. The examples of special stains are flagellar stain and Schaeffer-Fulton spore stain. Gram stain is the famous and world widely applied in research for differential staining. It was created by Hans Christian Gram in 1884 and used to distinguished four groups of organisms. The first group is Gram-positive organisms, whose cell walls retains crystal violet stain and give purple in colour. The second group is Gram-negative organisms, whose cell walls do not retain crystal violet and give red in colour. The third organism are Gram-variable, which unevenly stained and the last group is Gram-negative, which did not retain any colour or poorly stained. Gram stain is very important techniques as misinterpretation of this technique can lead to false result or delayed diagnosis of infectious disease. The Ziehl-Neelsen acid-fast stain is a stain to detect tuberculosis and leprosy-causing organisms of the genus Mycobacterium. Certain bacterial cell walls contain high concentration of dense ‘waxy’ lipids that prevent the penetration of water. Thus, the basic dye such as crystal violet is neglected for the acid-fast bacteria. Therefore, acid-fast stain uses procedure that forces dye through this nearly waterproof cell wall. Those that are “acid-fast” retain red carbol-fuchsin that resulted red in colour. Spore-forming bacteria are responsible for several serious diseases as well as one type of food poisoning. Endospore walls are very resistant to penetration of ordinary stains. Therefore, the Schaeffer-Fulton spore stain makes spore easier to see through microscope. The Endospore stain is very important especially to detect genus Clostridia; that responsible for gas gangrene, botulism and tetanus and genus Bacillus that cause the disease anthrax.
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PRACTICAL 1-BACTERIAL STAINING AND COLONY CHARACTERISTICS
INTRODUCTION
Just as different plants and animals have various morphologies or shapes, so do bacteria, both
microscopically and microscopically. The observing of bacteria colonies or the examination of plate
cultures are very important since bacteria were majorly first cultured on solid media such as agar
media. In addition, this technique is very important since different bacteria possesses different
morphology and might be used to differentiate between microorganisms in further test. In this
practical, 13 types of bacteria cultures were given and their morphology was observed such as size,
margin, elevation, colour, appearance and opacity.
The further step in order to examine the bacterial morphology is the staining technique. A
stain or dye is a molecule that can bind to a cellular structure and give it colour. Staining techniques
are very important as it can be used to investigate the structures, the chemical reaction towards the
cell and thus, categorise it. There are two types of dyes, the cationic or basic dyes and anionic dyes.
The examples of cationic dyes are crystal violet and malachite green and the anionic dyes are eosin
and picric acid. The staining techniques consist of three types; simple staining (used only one type of
dye) and differential staining (used more than one dye) and special staining. The examples for
differential staining are Gram stain, Ziehl-Neelsen acid-fast stain and negative stain. The examples
of special stains are flagellar stain and Schaeffer-Fulton spore stain.
Gram stain is the famous and world widely applied in research for differential staining. It was
created by Hans Christian Gram in 1884 and used to distinguished four groups of organisms. The
first group is Gram-positive organisms, whose cell walls retains crystal violet stain and give purple in
colour. The second group is Gram-negative organisms, whose cell walls do not retain crystal violet
and give red in colour. The third organism are Gram-variable, which unevenly stained and the last
group is Gram-negative, which did not retain any colour or poorly stained. Gram stain is very
important techniques as misinterpretation of this technique can lead to false result or delayed
diagnosis of infectious disease.
The Ziehl-Neelsen acid-fast stain is a stain to detect tuberculosis and leprosy-causing
organisms of the genus Mycobacterium. Certain bacterial cell walls contain high concentration of
dense ‘waxy’ lipids that prevent the penetration of water. Thus, the basic dye such as crystal violet is
neglected for the acid-fast bacteria. Therefore, acid-fast stain uses procedure that forces dye
through this nearly waterproof cell wall. Those that are “acid-fast” retain red carbol-fuchsin that
resulted red in colour.
Spore-forming bacteria are responsible for several serious diseases as well as one type of
food poisoning. Endospore walls are very resistant to penetration of ordinary stains. Therefore, the
Schaeffer-Fulton spore stain makes spore easier to see through microscope. The Endospore stain is
very important especially to detect genus Clostridia; that responsible for gas gangrene, botulism and
tetanus and genus Bacillus that cause the disease anthrax.
METHODS AND MATERIAL
METHODs-refer schedule
MATERIALS-13 pure cultures labelled A to M were prepared. (refer schedule)
I 1-2mm White Circular Filamentous Umbonate Opaque
J 1mm Creamy Circular Entire Umbonate Opaque
K 1-2mm Creamy Circular entire Convex Opaque
L
<1 mm Yellowish-orange
Circular Entire Raised Translucent
M
3 Green Irregular Entire Convex Translucent
B-GRAM STAINING
Bacteria Gram-positive/negative Shape
A Gram-negative ENTERIC ROD
B Gram-positive ROD
F Gram-positive ROD
G Gram-positive COCCI
I Gram-positive BRANCHED COCCI
M Gram-negative COMMA/ROD
K Gram-positive TETRACOCCI
C-SPORE STAINING D- ACID- FAST STAINING
DISCUSSION
An experiment was undergoing to examine the morphology of the culture based on the colony
characteristics and staining result. For the colony morphology several characteristics such as size,
colour, form/texture, margin, elevation and transparency were observed and recorded.
For the size of colonies, we can categorise it into 3 groups. The first group is the bacteria
that grew not more than 1mm. It was observed that only one bacterium that was bacteria L that
grew less than 1 mm. The second group is bacteria that grew within 1-5mm. From the experiment,
majority of bacteria grew within this group such as bacteria A, B, E, F, G, H, I, J, K and M. The third
group was bacteria that grew more than 5mm. It was observed those only 2 bacteria that located in
this group such as bacteria C and D. There are several physiochemical factors that affect growth of
bacteria. They are temperature, pH of media or environment, water activity/solutes and availability
of oxygen. Plus, the nutrition in media also influenced the growth of bacteria
For the colour characteristic, majority of bacteria have creamy in colour. The examples of
bacteria were A, C, D, F, G, J and K. There were2 bacteria that possessed white in colour. They were
bacteria B and I. They were only one bacterium that possessed reddish pink, grey, yellowish orange
and green in colour. They were bacteria E, H, L and M respectively. For the form/ texture
characteristic, we can observed that majority of bacteria such as bacteria A, B, C, E, F, G, I, J, K and L
have circular in form. The bacterium D had rhizoid in form. The bacterium H had round in form and
the bacterium had irregular in form.
Bacteria Spore staining
A NEGATIVE
B NEGATIVE
C POSITIVE
D POSITIVE
I NEGATIVE
L NEGATIVE
J NEGATIVE
K NEGATIVE
Bacteria Acid-Fast Staining
B NEGATIVE
I NEGATIVE
L POSITIVE
For the margin characteristic, majority of bacteria had entire margin. They were bacteria A,
E, F, H, J, K, L, and M. They were two bacteria that had undulate margin; bacteria C and D. The rest
have lobate and filamentous margin; bacteria B and I respectively. For the elevation characteristic,
most bacteria have raised elevation; bacteria B, F, G, H and L. They were two bacteria that have flat,
convex and umbonate elevation; bacteria C and D (flat), bacteria K and M (convex) and bacteria I and
J (umbonate). For the transparency or opacity characteristic, most bacteria were translucent. They
were bacteria A, B, C, D, F, H, L and M. The rest had opaque opacity; bacteria E, G, I, J and K.
For the Gram staining method, we can observe that bacteria B, F, G, I and K were Gram-
positive. This is because there were retained the purple in colour. Theoretically, the Gram stain
reaction is based on the amount or thickness of peptidoglycan found in the cell walls of the bacteria.
Gram-positive bacteria have many layers of peptidoglycan, which in turn holds molecules of techoic
acids. Techoid acid reacts with crystal violet and iodine used in this staining process. A complex of
crystal violet-iodine-techoic acid molecules form, which give purple/violet in colour and difficult to
remove these complexes. The cell wall of Gram-positive are quite thick (20-80nm) and consists of
between 60% and 80% peptidoglycan, which is extensively cross-linked in 3-D to form a thick
polymeric mesh. This statement proves that the complexes that form from the Gram-positive cell
wall difficult to remove eventhough alcohol are being used as the decolorizing agent.
The bacteria A and M were Gram-negative. Gram-negative bacteria have only one layer of
peptidoglycan with no techoic acid. So, when the decolorizing agent being applied, the alcohol
mixture (decolorizing agent) dissolves much of the polysaccharide outer layer, which further remove
the crystal violet primary stain from these cells. The Gram-negative resulted red in colour because
the reaction of the safranin red, the caounterstain. The colorless Gram-negative absorb the red
colour of the safranin. The Gram-positive did not react with safranin because it already holds the
crystal violet-iodine-techoic complex; make it stained darker of this complexes colour.
For the acid-fast technique, only 3 types of bacteria were examined; bacteria B, I and L. The
purpose of these staining is to detect the genus Mycobacterium and Nocardia species, the acid-fast
organisms that contain high concentrations of dense ‘waxy’ lipids known as mycolic acid. These
structures resist to the aqueous dye such as crystal violet. The result, only bacteria L was positive in
acid-fast stain. The positive acid-fast bacteria retain its bright red in colour and even resist
decolorization with acid alcohol. The acid fast bacteria are usually performed when the result in
Gram stain is in intermediate form; half violet and half red. It is called as gram-variable.
For the endosprore staining, only bacteria C and D gave the positive result. This indicates
that both bacteria have a spore. Bacterial endosprore are made up of genetic material, heat-
resistant enzymes, less water and a thick, waterproof outer protein called the spore coat. This spore
coat is the main indicator for these staining. The technique are same with the acid fast, it force dye
to enter through the spore coat. The malachite green (dye) act as primary stain and colour
everything with green. Thus, the only positive spore staining bacteria such as Bacillus and
Clostridium species remain green and the other vegetative cells, will be red in colour (red safranin
was used to act as counterstain and cannot penetrate the spore coat). But, safety precautions have
to take during undergo these technique. Certain vegetative cells such as Mycobacteria, dust and
debris often are not decolorized and will appear as green in colour too. Plus, there were also
exospores, the condition when the spore is fully formed and die of the vegetative cells that produce
it.
CONCLUSION
In a nutshell, different bacteria colonies produce difference morphologies. We can observe their
morphology in aspect of size, margin, elevation, opacity and many more. The Gram stain is used to
differentiate bacteria to its thickness of peptidoglycan. The uncertainty of the result in Gram stain
(usually happened in Gram-variable) can be identified with the application of acid-fast stain. The
uncertainty happened due to the presence of mycolic acid structure in cells. The bacteria that
produce spore or Endospore former can be identified using spore stain techniques.
QUESTION
1. Bacterial colonies with different colony characteristics growing on a microbial media
indicate the bacterial diversity in a sample.
Do you agree with the above statement? Give reasons to support your answer.
Yes. Because different bacteria give rise to different morphology and characteristic on the
media. The colony characteristics are important in order to differentiate between bacteria
and some can be useful in order to distinguish bacteria in superficial level of classification.
Plus, it is also the precursor step before different techniques such as staining and
biochemical test take place. Thus, the colony characteristics indicate the bacterial diversity
in a sample based on the reason that being state above.
2. The three staining methods above are differential staining methods. What is the principle
or theory of each method? Give examples how the three staining methods can help in the
identification of bacteria.
The Gram stain is the differential type of stain to detect the thickness of peptidoglycan on
bacteria. The bacteria that have thick peptidoglycan are said as Gram-positive bacteria while
the bacteria that have thin peptidoglycan are said as Gram-negative bacteria. The Gram-
stain techniques involve the series of several stain application. Firstly, the crystal violet
solution (primary stain) to a bacteria smear followed by applying an iodine solution (act as
mordant) follow with decolorization agent, the alcohol and then lastly, applies the safranin
solution (counterstain).
The crystal violet solution dissociates into CV+ and Cl- ions that eventually
penetrating through the wall of bacterial membrane. The CV+ reacts with negatively
charged structure of bacterial cells that causes a purple in colour. The applying of iodine that
creates large complexes of CVI that hard to breakdown. The complexes are formed within
the cytoplasm and outer layer of cells. The crucial step in Gram-stain is decolorization with
alcohol. The inaccuracy of doing step can cause the false interpretation about the properties
of bacteria. The Gram-negative bacteria have thin peptidoglycan layer beneath the outer
layer of cells. Thus, the decolorizing agent disrupts the outer layer of cell and allows the CVI
complexes to be wash away. The Gram-positive have thick, high cross-linked peptidoglycans
that have ability to trap the CVI complexes and withstand with the alcohol. The counterstain
agent (positively charged dye safranin) then being reacts with the Gram-negative
peptidoglycan that already loses its purple in colour. Therefore, the colour for Gram-positive
bacteria is purple and the colour for Gram-negative bacteria is red.
In certain cases, some bacteria produce an intermediate colour of Gram-staining.
They give a pattern of purple and red in colour. This is what we called as Gram-variable. This
is because the presence of waxy peptidoglycan called mycolic acid in peptidoglycan
structure. Therefore, a further staining such as acid-fast staining is important to correct this
problem.
The Ziehl-Neelsen acid-fast stain is a stain to detect tuberculosis and leprosy-causing
organisms of the genus Mycobacterium. Certain bacterial cell walls contain high
concentration of dense ‘waxy’ lipids that prevent the penetration of water. Thus, the basic
dye such as crystal violet is neglected for the acid-fast bacteria. Therefore, acid-fast stain
uses procedure that forces dye through this nearly waterproof cell wall. Those that are
“acid-fast” retain red carbol-fuchsin that resulted red in colour.
Spore-forming bacteria are responsible for several serious diseases as well as one
type of food poisoning. Bacterial endosprore are made up of genetic material, heat-resistant
enzymes, less water and a thick, waterproof outer protein called the spore coat. This spore
coat is the main indicator for these staining. The spore staining technique is same with the
acid fast, it force dye to enter through the spore coat. The malachite green (dye) act as
primary stain and colour everything with green. Thus, the only positive spore staining
bacteria such as Bacillus and Clostridium species remain green and the other vegetative
cells, will be red in colour (red safranin was used to act as counterstain and cannot penetrate
the spore coat). But, safeties precautions have to take during undergo this technique.
Certain vegetative cells such as Mycobacteria, dust and debris often are not decolorized and
will appear as green in colour too. Plus, there were also exospores, the condition when the
spore is fully formed and die of the vegetative cells that produce it.
REFERENCES
James G. Cappucino, N. S. (1999). Microbiology-A Laboratory Manual (5th Edition ed.). 2725 Sand Hill Road, Menlo Park California: Benjamin/Cummings Science
Robert A. Pollack, W. M., Lorraine Findlay, R. Ronald Modesto. ( 2002). Laboratory
Exercises in Microbiology. Rosewood Drive, Danvers, MA: John Wiley & Sons, Inc.
Black, J. G. (2002). Microbiology: Principles and Explorations (5th Edition ed.). New
York, USA: John Wiley & Sons. Benson, H. J. (2002). Microbiological Applications: Laboratory Manual in General
Microbiology (8th ed.). NY, USA: McGraw-Hill Companies. Donald Breakwell, C. W., Bryan MacDonald, Kyle Smith & Richard Robison. (2007).
Colony Morphology Protocol Retrieved 15 November, 2012, from http://www.microbelibrary.org/component/resource/laboratory-test/3136-colony-morphology-protocol
Marise A. Hussey, A. Z. (2008). Acid-Fast Stain Protocols Retrieved 15 November,
2012, from http://www.microbelibrary.org/component/resource/laboratory-test/2870-acid-fast-stain-protocols
Marise A. Hussey, A. Z. (2007). Endospore Stain Protocol, from
PRACTICAL 2-POUR PLATING AND SPREAD PLATING TECHNIQUES FOR THE
ENUMERATION OF BACTERIA
INTRODUCTION
Instead of observing and examined the bacteria culture, obtaining the number of bacteria present in
sample such as water, milk and food are also important. Many (microbiologist and other expertises)
believe that we be able to determine the number of bacteria that are present in a given unit of
volume. A quantitative plating method or standard plating method (SPC) is a technique that is
widely used to enumerate the number of bacteria. SPC is done based on facts that only viable
bacterium will divide and form a visible colony on an agar plate.
In addition, SPC technique also involve procedure of diluting the organisms with a series of
sterile distilled water/ distilled water before undergo either streaking or pouring plate techniques.
This is very important because it is irrelevant or difficult to count the colony that has too many or to
less in number. In addition, a single bacterium is deposited on an agar plate, will divide to form a
colony. Each bacterium represents a colony-forming unit (CFU). The acceptable range for counting
and recording is between 30-300 colonies in one agar plate. The serial dilutions start with adding of
1 ml of bacteria suspension or sample to 9 ml of distilled water to makes a 10-1 dilution. Then, add
1ml of 10-1 dilution to 9 ml of distilled water to make a 10-2 dilution. These steps are keeping
repeating until you obtained the 10-3, 10-4, 10-5 and many more. This is also called a dilution factor.
The spread plate technique is a consequence steps after dilution series technique. The
diluted sample is first placed on the centre of solid agar before being spread using the sterile, bent
glass rod (usually called as hockey stick). This technique develops the colonies on the surface of agar
after appropriate time and condition of incubation. Instead of spread plate, pour plate technique is
also used in order to enumerate the bacteria. 1 ml of diluted sample (from serial dilution) is added to
9 ml of melted agar and the mix is undergo an incubation period. The colonies were formed in the
middle of the agar.
The streak plate technique is the method to isolate colonies from large numbers of different
bacteria. The streaking is done when a single, pure colony was obtained and isolated from other
colonies. This technique is very important as obtaining a single, pure colony can be used for further
steps such as biochemical test or manipulate in for genetic engineering purpose.
MATERIAL AND METHODS
MATERIAL
A bottle of water sample from 12th Residential College, University of Malaya
A broth containing mixed culture of Micrococcus luteus and Serratia sp.
METHODS-(refer schedule)
RESULTS
1. SERIAL DILUTION-SPREAD PLATE TECHNIQUE
DILUTION FACTOR
10-1 10-2 10-3 10-4 10-5 10-6
CFU TNTC TNTC TNTC TNTC 305 33
The concentration of bacteria : 33 X 106 = 3.3 X 107 cfu/0.1 ml
= 3.3 x 108 cfu/ml
2. SERIAL DILUTION-POUR PLATE TECHNIQUE
DILUTION FACTOR
10-1 10-2 10-3 10-4 10-5 10-6
CFU TNTC TNTC TNTC 304 135 40
Ratio between the number of colonies in 10-5 and 10-6 dilution factor :
= 2.96
The concentration of bacteria : 135 x 105 = 1.35 x 107 cfu/ml.
3. DILUTION STREAKING TECHNIQUE
Colour of colony produced
Single colony produced
Morphology
Size Margin Elevation Appearance
Red 11 3mm Entire Convex Circular
DISCUSSION
In the spread plate techniques, 6 dilutions factors; from 10-1 till 10-6 are prepared and the bacteria in
each dilution factor were incubate on agar plate media for overnight at 37oC. For the dilution factor
10-1 till 10-4, the colonies formed on the media are too many and the countable become unreliable.
Thus, these colonies are marked as TNTC, an acronym for too numerous to count. Only colony in
dilution factor 10-5 and 10-6 can be count. For calculating the concentration of bacteria for the
spread plate technique, we choose the number of colony in dilution factor 10-6 that is 33 and did
not choose from the dilution factor 10-5 because the best range to choose is from 30-300.
Therefore, the concentration number in this technique is 3.3 x 108 cfu/ml.
In pour plating technique, the same dilution factors were used and incubate on same media
and period of incubation like the spread plating technique. For dilution factor of 10-1 till 10-3, the
colonies formed are marked as TNTC. They are three dilution factors that have reliable number of
count; 10-4 has 304 numbers of colonies, 10-5 has 135 numbers of colonies and 10-6 has 40 numbers
of colonies. The number of colonies in 10-5 and 10-6 located in the range 30-300 colonies. Therefore,
the number of colonies in highest division has to divide to the number of colonies in lowest dilution
to get the ratio between them. If the ratio obtained is 2 and below, the average between them are
calculated. But, if the ratio is higher than 2, the number of colonies in lowest dilution will be chosen.
From the calculation, the ratio obtained is 2.96. Therefore, the number of colonies in 10-5 being
chose and the concentration of bacteria for the pour plate technique is 1.35 x 107 cfu/ml.
Based on the bacterial concentration for both techniques, we can see that the spread plate
technique has higher number of bacterial concentration compared to the pour plate technique. They
are several reasons of this situation. First, the aerobic bacteria cannot grow well in the pour plate
technique as this technique involves the forming of colony under the surface of agar. It is suitable to
identify the facultative aerobic and anaerobic bacteria only. In addition, the high temperature
involved during pour the hot molten agar eventually kills the bacteria as the bacteria experience
heat stress and cannot withstand with high temperature.
In dilution streaking, two different bacteria were mixed in a broth. Thus, the streaking
technique is applied to differentiate between these two bacteria. The result is only a type of colony
formed on the agar. The colonies have 3mm in size, entire margin, convex elevation and circular
appearance. Based on the morphology observed, we can conclude that the bacteria for the colony is
from Serratia sp. However, the other type of colony did not form on the agar. The might be
possibilities of this situation. Maybe, the period of incubation is too short and the bacteria cannot
grow on nutrient agar.
CONCLUSION
The spread plate and pour plate techniques are example for enumerating of bacteria. Before
undergo this two techniques, a serial of dilution series have to be done in order to decrease the
concentration of bacteria from original sample, make it reliable to count. The streaking technique
can be used to differentiate and isolate the mix bacteria in a sample. However, the accurate result
cannot be achieved if we did not consistently applying the aseptic technique while undergo the
experiment.
QUESTION
1. Compare and contrast between these two enumerations techniques. Discuss both the strengths
and weaknesses of these techniques.
POUR PLATE SPREAD PLATE
SIMILIARITIES
Both need a period of incubation with appropriate time and temperature.
Both used a petri dish as a medium of technique.
Both used organisms or bacteria to enumerate.
Both have to undergo a serial dilution technique. DIFFERENCES
Colonies formed below surface of agar media (surface/subsurface colonies).
Colonies formed on a surface of agar media (surface colonies).
Colonies are restricted in respective size. Colonies have a capability to increase in size. The detection limit is 1.0 ml. The detection limit is 0.1 ml. The bacteria are experiencing heat stress. The bacteria are experiencing physical stress. Difficult to isolate. Easy to isolate. The minimum cfu limit is 1cfu/ml. The minimum cfu limit is 10cfu/ml. The first thing added is the bacterial broth. The first thing added is the melted agar.
The advantages of having the pour plate technique are suitable for quantification of colonies in solid
medium. It allows the growth and quantification of macrophiles bacteria. Means that, if we want to
undergo the experiment involving the anaerobic bacteria, this technique is more preferable. In
addition, these techniques can reduce the total of bacteria count as highly aerobic and heat sensitive
bacteria cannot grow well under the surface of agar media. The disadvantages of this technique are
it interrupts other neighbour colonies as the selected colonies must be digging out in agar. In
addition, the microbes must withstand the agar temperature due to heat stress.
The advantages of having the spread plate technique are the cultures are never exposed to
45oC melted agar temperature. Therefore, the bacteria did not experiencing heat stress. In addition,
many viable microbes can be culture using this technique. The disadvantages of these techniques
are more microbes can be presence in the media. Thus, it is very difficult for us to specify to the
interest bacteria. In addition, they are possibilities of the bacteria to die due to physical stress during
spread.
2. Although dilution streaking can separate different bacterial isolates, sometimes the colonies
observed after dilution streaking are still mixed cultures. What can we do to purify them?
In order to purify them, we have to undergo the streak plate in a new plate for 3 times, the optimum
number for repeating streaking. The concept of streaking is done in such a way as to ‘thin out’ the
microbes so that, eventually only one bacterial cell at a time is placed on the plate, well separated
from the others. Therefore, keep re-do the streaking will eventually loosen the ‘clump’ of bacteria
and thus, obtain the pure, single colony.
APPENDICES
Figure 1: The colonies formed on nutrient agar using the spread plate techniques
Figure 2: A type of colony formed using
streaking technique. Only one type
bacterium grow on agar.
REFERENCES
Robert A. Pollack, W. M., Lorraine Findlay, R. Ronald Modesto. ( 2002). Laboratory Exercises in Microbiology. Rosewood Drive, Danvers, MA: John Wiley & Sons, Inc.
Benson, H. J. (2002). Microbiological Applications: Laboratory Manual in General
Black, J. G. (2002). Microbiology: Principles and Explorations (5th Edition ed.). New York, USA: John Wiley & Sons.
James G. Cappucino, N. S. (1999). Microbiology-A Laboratory Manual (5th Edition ed.). 2725 Sand Hill Road, Menlo Park California: Benjamin/Cummings Science
PRACTICAL 3- MOST PROBABLE NUMBER (MPN) AND MEMBRANE
FILTRATION TECHNIQUES FOR THE ENUMERATION OF
COLIFORM
INTRODUCTION
Water is one of the sources of numerous kinds of bacteria such as autotrophs and saprophytic
heterotrophs bacteria. Based on this fact, we have to consider the source of water in order to know
the organisms that exist for various purpose such as to drink and to detect pathogens that can our
life. Thus, the Most Probable Number and Membrane Filtration Techniques can be used to
enumerate the bacteria or specifically to detect a coliform in water. Coliform is facultative, gram
negative and non-spore-forming rod anaerobes that ferment lactose to produce gas. Based on this
definition, bacteria such as Escherichia coli and Enterobacter aerogenes are examples of coliform.
Most Probable Number technique is applied to eliminate the problems such as organisms
that present is too irrelevant to undergo standard plate count and condition where zero growth on
agar after incubate. With this method, we can observe the sample and estimates the number of cells
using the table of MPN. A typical MPN consists of three or five tubes that consist of an inverted tube
(usually called as Durham tubes), the broth or any detection agent and the microbial culture. Those
that contain an organism will display growth by producing gas bubbles and/or becoming cloudy or
change the broth’s original condition after incubate. The number of organisms in the original culture
is estimated from a MPN table, a table that based on the statistical probabilities. The broth used in
this experiment is MacConkey broth. The MacConkey broth contains lactose that facilitates the
lactose-fermentor bacteria to ferment bacteria. Plus, the composition of bromocresol purple as an
indicator for acid production. All these criteria are the best way to enumerate the coliform.
Membrane filtration technique is a technique of filtration of unwanted organisms from pass
through the pore and thin disk called membrane filter. It is usually made from nitrocellulose and has
its own pore sizes from 25µm to less than 0.025µm. The membrane filter has many uses. It used to
sterilize media, pharmaceutical products, and vitamins, in manufacturing vaccines and in sampling
microbes in air and water.
The haemocytometer or hemocytometer (other state as counting chamber) is the device
created by Louis-Charles Malassez, a French anatomist and histologist. The original purpose of this
device is to count the blood cells. Nowadays, it had been modified and almost all organisms can be
count using this device. The haemocytometer is one of the techniques that count directly the
bacteria under the microscope. It consists of a cover glass and a counting grid that consists of several
1 mm2 grids and being subdivided into 3 small grids; 0.0625 mm2, 0.05 mm2 and 0.04 mm2. There are
few things to consider in using this device. They are type of counting chamber, the cover glass used,
moving cells condition and the microscope objective lens.
METHODS AND RESULT
METHODS-(Refer schedule)
RESULT
1. MOST PROBABLE NUMBER TECHNIQUE
DILUTION FACTOR TEST TUBE
10-4 10-5 10-6
1 + + - 2 + + - 3 + + -
Number of tubes with positive result 3 3 0 (+=refer to positive result) and (-=refer to negative result)
The MPN code by referring the Hoskin’s table: 24
Thus, the bacterial concentration is: 24 X 105=2.4 X 106 MPN/ml
2. MEMBRANE FILTRATION TECHNIQUE
Dilutions Colony-forming Unit (CFU) on plate
Colour of colony
10-3 TNTC Pink
10-4 58 Pink
10-5 1 Pink
The bacterial concentration: 58 X 104 = 5.8 X 105 cfu/ml
DISCUSSION
From the result recorded above, the test tubes in dilution factor of 10-4 and 10-5 show the positive
result. The positive result means that the MacConkey broth changed the colour from purple to
yellow and they were gas bubble in Durham tube. This indicates that the coliform is present since
the criterion of coliform is facultative anaerobes that ferment lactose and yield gas and acidic
condition. If the result is either colour change or gas bubble produce, it still counts as negative. But,
there are some difficulties during undergo this technique. Not all group obtained the acquired result.
This is because there will be some contamination occur during undergo the test due to poor aseptic
technique being applied. The result obtained is 330 and by referring the Hoskin’s table, the 330 key
codes that we got in MPN technique is equal to 24. Thus, the bacterial concentration is 2.4 X 106
MPN/ml.
For the membrane filtration technique, all the MacConkey agar plates grew the colonies
after a period of incubation. We only count for the colonies that red in colour and not the white in
colour. This is because the lactose fermentor will utilize lactose (one of the composition in
MacConkey broth agar) and lowers the pH of agar below 6.8 and result in the red in colour.
Meanwhile, for non-lactose fermentor, it utilizes peptone and produce ammonia that increase the
pH of agar and result in the white or colorless. For the plate with dilution factor of 10-3, they were
numerous and uncountable number of colonies. The plate is marked as too numerous to count
(TNTC), standard term for the plate that have unacceptable number (too many) of colonies. For the
plate with dilution factor of 10-4, there were 58 colonies form and for the dilution factor of 10-5, they
were a single colony produced. Therefore, the bacterial concentration is 5.8 X 105 cfu/ml.
In addition, we were given the improved Neubaeur haemocytometer to carry out the direct
bacterial count. We had to control the amount of light in order to see the grid before undergo the
bacterial count. The improper illumination will cause the inaccuracy to count the bacteria.
CONCLUSION
There are several of techniques to enumerate the coliform in water sample. In this practical, we had
been introduced to Most Probable Number technique and Membrane Filtration technique. Both
techniques have its own advantages and disadvantages; depend on the nature of the sample being
used. In addition, the aseptic technique has to apply in both techniques in order to avoid
contaminations that eventually produce the inaccuracy result. The haemocytometer is one of the
simple devices that give us a direct bacteria count without having the agent media or incubation
period. It is simple method but, without proper technique, it will be difficult for us to obtain the
result.
QUESTIONS
1. Compared and contrast between these two enumerations techniques. Discuss both the
strengths and weaknesses of these techniques.
MOST PROBABLE NUMBER MEMBRANE FILTRATION TECHNIQUE
SIMILIARITIES
Both need a period of incubation with appropriate time and temperature.
Both are techniques to enumerate coliforms in water sample.
Both used organisms or bacteria to enumerate.
Both have to undergo a serial dilution technique.
DIFFERENCES
The MPN used MacConkey broth as a media. The MFT used MacConkey solid agar as a media.
The coliform formed is in the broth and cannot undergo a direct bacteria count.
The coliform formed on the solid agar and can undergo a direct counting.
All size of bacteria can grow in the broth. Only the selective bacteria that below the pore size can grow in solid agar.
The MPN used a Durham tube to collect the gas formed.
The MFT did not collect gas.
The MPN did not use any filter. The MFT uses a membrane filter.
The MPN technique is quite difficult to be done. The MFT technique is much more simple and precise.
The advantages of using Most Probable Number techniques are this method result in
uniform result, therefore easier for us to enumerate the coliforms. In addition, it is only detect the
viable organisms based on the broth change and gas produced. Plus, it can enumerate many
coliforms as they are no restriction size of bacteria in this technique. The advantages of MPN
technique are large number of replication is needed to narrow the confidence intervals. Plus, it is
only involve the estimation of coliforms, and only produce 95% of certainty by using the statistical
possibilities table.
The advantages of Membrane Filtration technique are it can be used on large sample
volumes as it is not easy to clog. Plus, it save time and less steps applied. Furthermore, the coliforms
can be directly counted and can be specifically used to target bacteria as it purchase with the
membrane filter in order to remove unwanted microbes and debris. Plus, it can be autoclaved, so
that it can be used it back in future. In the left hand, the membrane of MFT cannot filter viruses and
other mycoplasmas. Plus, the MFT may absorb relatively large amounts of the filtrate and may
introduce metallic ions into the filtrate.
2. Show how you calculate the volume of the haemocytometer chamber. Discuss the difficulties
faced when using the haemocytometer and how we can overcome these difficulties?
The haemocytometer chamber used in diagram above is the improved Neubaeur haemocytometer.
We have to know what is chamber used because different chambers have different grids. The
improved Neubaeur haemocytometer consists of a large square of 1mm2 (the blackened line). To
acquire the volume of the square, we have to multiply with 0.1mm as it is the depth of the cover
glass. Thus, the volume of the large square is 0.1ml or 100µl. The large square just now is divided
into 3 different squares with different volume. Region 2 consists of small squares that have an area
of 0.0625mm2 (0.25mmx0.25mm) or 6.25µl of volume for each. Region 3 consists of small squares
that have an area of 0.04mm2 (0.2mmx0.2mm) or 4µl of volume for each. While the main region or
Region 1 consists of squares that have an area of 0.0025mm2 (0.05mmx0.05mm) or 0.25µl in volume
for each. The overall volume of the haemocytometer is 900µl or 0.9ml. To acquire the
concentrations of cells in original sample, we can express using this formula:
Even though the haemocytometer is simple and direct, there still few errors and difficulties
that occur by using this technique. Firstly, the suspension under the chamber is not uniform. There
are possibilities of cell clumping together and produce inaccuracy result. Therefore, to overcome this
problem the sample has to be totally mixed before sample.
Second, the chamber must be completely clean. Improper filling will cause the chamber
become dirty and make the suspension did not flow smoothly in on the chamber. To overcome this
problem, the chamber and cover slip has to be clean with distilled water before applying the
ethanol. Next, wipe it with a Kim wipe.
The third difficulties happen when the cell is located on the border of the gridlines. Thus, it
will be hard for us to determine whether it should be counted or not. Therefore, to overcome this
problem, we should be consistent with our counting. We have to assume that the bacteria that
located on the line should not be counted. Thus, a consistent result can be obtained.
APPENDICE
Figure 1: The filter membrane technique, a technique to test
for the presence of coliform
REFERENCES
Robert A. Pollack, W. M., Lorraine Findlay, R. Ronald Modesto. ( 2002). Laboratory Exercises in Microbiology. Rosewood Drive, Danvers, MA: John Wiley & Sons, Inc.
Benson, H. J. (2002). Microbiological Applications: Laboratory Manual in General
Black, J. G. (2002). Microbiology: Principles and Explorations (5th Edition ed.). New York, USA: John Wiley & Sons.
James G. Cappucino, N. S. (1999). Microbiology-A Laboratory Manual (5th Edition ed.). 2725 Sand Hill Road, Menlo Park California: Benjamin/Cummings Science
MacConkey Broth. Retrieved 19 November, 2012, from
The concentration of hydrogen and oxygen gas then being reacted with catalyst (Palladiumised
alumina) to form water.
2 H2 + O2 + [Catalyst] = 2 H2O + [Catalyst]
All these 3 reactions keep the jar free from oxygen.
3. Discuss the theory or principle behind the NO2 test in the API test strip.
Sometimes, the 7-digit profile is not a valid confirmation result to detect the bacteria. Further tests
should be included in order to improvise the result. The reduction of nitrates to nitrites and nitrogen
gas are done in glucose oxidation/fermentation microtube. This is because the potassium nitrate is
being located in GLU tube. The NIT 1 and NIT 2 then added in GLU tube. The NIT 1 and NIT 2 are
sulfanilic acid and alpha-naphthylamine. The bacteria that have an enzyme nitrate reductase will
reduce nitrate to nitrite.The nitrite react with these two acids to form p-Sulfobenzene-azo α-
Naphthylamine, a water soluble red azo dye. Thus the red in colour formed is the positive result for
detect the enzyme.
4. By looking at the design of the Enterotube below, discuss its weakness and strengths.
The Enterotube system is used to identify enteric pathway or organisms that cause intestinal diseases
such as typhoid and paratyphoid fevers, shigellosis, gastroenteritis and other food poisoning cause
bacteria. The advantages of using this system are it combines several of tests into one test. In this
aspect, it saves time as well as the number of inoculation used. In addition, it is convenient and easy
to use as the size of the Enterotube is small and easy to carry handle.
The disadvantage of the Enterotube system is the result achieved is not totally accurate. The culture
is not well distributed in all tube. Thus, it might affect the result as the initial tube will have higher
concentration of bacteria compared to the end of the tube.
APPENDICES
Figure 1: The decarboxylase test
result for bacteria in
culture Klb
Figure 2: The oxidase test. Noted
that the dark blue formed
indicate the positive result
Figure 3: The Catalase
test. The positive
indicator show
the bubbles
formed.
Figure 4: The coagulase
Test. The
precipitation
indicate the
positive result
Figure 5: The haemolysis of blood agar and chocolate agar in anaerobic condition.
Figure 6: The API 20 E strip test result.
REFERENCES
Robert A. Pollack, W. M., Lorraine Findlay, R. Ronald Modesto. ( 2002). Laboratory Exercises in Microbiology. Rosewood Drive, Danvers, MA: John Wiley & Sons, Inc.
Benson, H. J. (2002). Microbiological Applications: Laboratory Manual in General
Black, J. G. (2002). Microbiology: Principles and Explorations (5th Edition ed.). New York, USA: John Wiley & Sons.
James G. Cappucino, N. S. (1999). Microbiology-A Laboratory Manual (5th Edition ed.). 2725 Sand Hill Road, Menlo Park California: Benjamin/Cummings Science
Triple Sugar Iron. Retrieved 21 November, 2012, from
Cathcart, P. S. L. (2010). Oxidase Test Protocol Retrieved 21 November, 2012, from http://www.microbelibrary.org/index.php/library/laboratory-test/3229-oxidase-test-protocol
Katz, D. S. (2010). Coagulase Test Protocol Retrieved 21 November, 2012, from http://www.microbelibrary.org/library/laboratory-test/3220-coagulase-test-protocol
Frankhauser, D. B. (2001). Triple Sugar Iron Agar and Its Use Retrieved 21 November, 2012, from http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Triple_Sugar_Iron/TSI_Use.htm
Citrate Utilization.). Retrieved 21 November, 2012, from http://web2.uwindsor.ca/courses/biology/fackrell/Methods/citrate.htm
Laboratory Identification of Vibrio Cholerea.). Retrieved 21 November, 2012, from https://docs.google.com/viewer?a=v&q=cache:opNGxEEc9fAJ:www.cdc.gov/cholera/pdf/laboratory-methods-for-the-diagnosis-of-vibrio-cholerae-chapter-6.pdf+vibro+cholare+on+dextrose+decarboxylase&hl=en&gl=my&pid=bl&srcid=ADGEESiZqRNj7Gly8No5Fd0SnyqVl_U_qcXuS27_QkBARVjwmqqXRbPcQcQ35wJzr8jJLio4it0Gtsce9m_0kkdhYBmO9g-E1HeiMni3wJ5_2b_aY2jFqCt2H0vlpkoKlC0AJCA05mCd&sig=AHIEtbQAwIkXhQzVwLNtO0woB9C3fyQaDQ