compilation of the bacteriology practical report

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)

A -Escherichia coli

B -Bacillus subtilis

C -Bacillus megaterium

D -Bacillus cereus

E -Serratia sp.

F -Staphylococcus aureus

G -Staphylococcus epidermidis

H -Salmonella typhi

I -Streptomyces griseus

J -Pseudomonas sp.

K-Micrococcus luteus

L -Nocardia sp.

M-Vibrio sp.

RESULT

A-COLONY MORPHOLOGY

Bacteria Size (mm) Colour Form/texture Margin Elevation Transparency

A 3 Creamy white

Circular Entire Convex Translucent

B 1 White Circular Lobate Raised Translucent

C 6 Cream Circular Undulate Flat Translucent

D 10 Creamy White

Rhizoid Undulate Flat Translucent

E 2 Reddish pink

Circular Entire Umbonate Opaque

F 3 Cream Circular Entire Raised Translucent

G 3 Cream Circular Undulate Raised Opaque

H

2-3 mm Grey Round Entire Raised Translucent

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

http://www.microbelibrary.org/library/laboratory-test/3112-endospore-stain-protocol

Ziehl–Neelsen stain. (2012). Retrieved 16 November, 2012, from

http://en.wikipedia.org/wiki/Ziehl%E2%80%93Neelsen_stain

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

Microbiology (8th ed.). NY, USA: McGraw-Hill Companies.

Black, J. G. (2002). Microbiology: Principles and Explorations (5th Edition ed.). New York, USA: John Wiley & Sons.


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






MacConkey Broth. Retrieved 19 November, 2012, from

https://docs.google.com/a/siswa.um.edu.my/viewer?a=v&q=cache:DGEWvKjDVDkJ:www.mastgrp.com/Dehydrated%2520culture%2520media/InfoSheet/DM/DM150%2520MacConkey%2520Broth.pdf+durham+tubes+with+macconkey+broth&hl=en&gl=my&pid=bl&srcid=ADGEESgklBwtvYxjDkya56zwWfBN3Pr7K2Pfv1WMflCdrGtT1wkHo-S9Y1ivyOzvuTEuUxuFMUgWN7yjLuc4PXDZ218fSVZjGZ3bbhYoHaO2sgeQYoCkzOREeoYxoWGdkT5muD1Fs4sF&sig=AHIEtbSnOQBeOb2WE-u1i4Rg817SsoSbbg

The hemocytometer (counting chamber). (2010). Retrieved 19 November, 2012,

from http://www.microbehunter.com/2010/06/27/the-hemocytometer-counting-chamber/

Hemocytometer. (2012). Retrieved 19 November, 2012, from http://en.wikipedia.org/wiki/Hemocytometer

MacConkey agar. (2012). Retrieved 20 November, 2012, from http://en.wikipedia.org/wiki/MacConkey_agar

Total Coliform Testing Procedures. Retrieved 20 November, 2012, from

http://water.me.vccs.edu/courses/env149/coliform.htm Membrane Filter Technique. (2012). Retrieved 20 November, 2012, from

http://www.pall.com/main/Laboratory/Literature-Library-Details.page?id=7290 Kirk, L. OVERVIEW OF MOST PROBABLE NUMBER COUNT METHODOLOGY

Retrieved 20 November, 2012, from http://filebox.vt.edu/users/chagedor/biol_4684/Methods/mpn1.html

Most probable number. (2012). Retrieved 20 November, 2012, from

http://en.wikipedia.org/wiki/Most_probable_number

PRACTICAL 4-BACTERIAL IDENTIFICATION USING BIOCHEMICAL TESTS

INTRODUCTION

Instead of knowing the morphology of the bacteria, the Gram classification and others, they are

many tests that being invented by scientist in order to seek out the identity or the other word to

gain seek out the extra ‘profile’ of certain bacteria. Plus, it also being used to identify and thus

classifying the unknown bacteria. One of the examples is the biochemical tests. The biochemical test

or usually known as ‘miscellaneous tests’ involve tests such as Indole production test, Catalase test,

Oxidase test, Citrate utilization test, Hydrogen sulphite test, Nitrate reduction test and many more.

In this practical, we will use several biochemical tests in order to familiar with it.

The first biochemical test that being practised is IMViC test. IMViC test is the combination of

Indole, Methyl Red, Voges-Proskauer and Citrate utilization tests. The purpose of Indole test is to

identify the bacteria that have the presence of set enzymes that convert typtophan to indole. The

bacteria are inoculated in the test tube a medium containing the tryptophan, a type of amino acid.

The organisms that have that particular enzyme will breakdown the tryptophan into indole. The

Methyl-red is the test of detecting the presence of acid condition produced by the bacteria. The

bacteria are cultured in MR-VP broth with the methyl-red indicator being added. The presence of

acid causes the colour of indicator change. The Voges-Proskauer is the test to detect the presence of

enzyme cytochrome oxidase in bacteria. The bacteria are cultured in MR-VP broth with alpha

naphtol and potassium hydroxide-creatine being added. If the bacteria contain this enzyme, it will

change the original colour of the broth to pink in colour. The Citrate utilization is the test to detect

the presence of permease complex, an enzyme that transports citrate into the cell. The bacteria are

inoculated into citrate agar medium in which citrate is the sole carbon source. The blue colour will

form, resulting of the positive result of this test.

Instead of IMViC test, the Oxidase test also is being carried out. The purpose of this test is to

detect the presence of cyctochrome oxidase in the certain bacteria. Two or three drops of an

oxidase test reagent are added to the bacteria on a piece of filter paper. The positive result of this

result occur when the reagent change it colour to blue, purple or black.

The third test that being carried out is the Catalase test. The nature of this test is to identify

the presence of catalase. The hydrogen peroxide is dropped on the culture (some culture was placed

on a glass slide). The formation of bubbles proves that the catalase oxidises hydrogen peroxide to

oxygen gas and water.

The Coagulase test is carried out to identify bacteria that produce enzyme coagulase, a

bacterial enzyme that accelerates the coagulation of blood. A drop of plasma is dropped onto a clean

glass slide. Then, the drop is mixed with the inoculating loop that contains culture. The precipitation

proves that the bacteria have coagulase enzyme.

TSI agar is a combination of lactose, sucrose and a small amount of glucose with the addition

of iron element such as ferrous sulphate and a pH indicator such as phenol red. The purpose of the

creation of this agar is to detect the bacteria that have ability to ferment carbohydrates and reduce

sulphur.

Decarboxylase test is the test to detect the presence of various decarboxylase enzymes in

bacteria. The Moeller Decarboxylase broth is the best broth to detect the presenceness of arginine,

lysine and ornithine decarboxylase enzymes and thus differentiate the genus Enterobacteriaceae

from others.

String test is test to differentiate Vibro species from other species. Sodium deoxycholate, a

detergent that used has the ability to lyse Gram-negative organisms. When cell are lysed, DNA is

released into the suspending medium making it very viscous and able to form ‘strings of DNA’ when

touched with a loop that is raised from the surface of the liquid. The positive result achieved when

string of DNA formed.

They are also a system that contain many tests arise from a single inoculation. The example

of this system is Analytical Profile Index. The API 20 E strip consists of 20 microtubes containing 20

different dehydrated substrates. The advantages of these systems are save cost and space and

incubation plus provide an efficient and reliable means of making positive identification of infectious

organisms. The 20 API E is a standardized identification system for Enterobacteriaceae and other

non-fastidious Gram-negative rods.

METHOD AND MATERIAL

METHOD-(refer schedule)

MATERIAL-12 bacteria cultures were prepared (refer schedule).

A: Escherichia coli

E: Serratia sp.

F: Staphylococcus aureus G: Staph. epidermidis

H: Salmonella typhi

I: Streptomyces griseus

J: Pseudomonas sp.

K: Micrococcus luteus

N: Streptococcus faecalis

Klb: Klebsiella sp.

MC: Vibrio cholerae

MV: Vibrio parahaemolyticus

RESULT:

A. IMViC Test

BACTERIA INDOLE TEST METHYL-RED TEST

VOGES-PROSKAUER TEST

CITRATE TEST

A Negative Positive Positive Negative

E Negative Negative Positive Positive

H Negative Positive Negative Negative

J Negative Negative Negative Positive

Klb Negative Negative Positive Positive

B. Oxidase Test

BACTERIA RESULT

A Negative J Positive K Negative

C. Catalase Test

BACTERIA RESULT

F Positive

G Positive

J Positive

K Positive

N Negative

D. Haemolysis of blood and chocolate agar in two different anaerobic environment

E. Coagulase Test

F. Triple Sugar Iron Test

BACTERIA OBSERVATION

CANDLE LIGHT JAR ANAEROBIC JAR

BLOOD AGAR CHOCOLATE AGAR

BLOOD AGAR CHOCOLATE AGAR

F Clear zone Semi clear zone Semi clear zone Dark, semi clear zone

G - Greenish, semi clear zone

- Clear zone

J Dark, semi clear zone

- Dark, semi clear zone

BACTERIA RESULT

F Positive

G Positive

K Positive

BACTERIA OBSERVATION

Slant condition Butt condition A Yellow in colour Breakage with yellow in colour

B Reddish pink in colour No breakage with reddish pink in colour

E Yellow in colour Breakage with yellow in colour

J Reddish in colour No breakage with reddish in colour

Klb Yellow in colour Breakage with yellow in colour

MC Yellow in colour No breakage with yellow in colour

MV No change observed. No change observed.

G. Decarboxylase test

H. String test

BACTERIA A F H J MV

STRING FORMATION Negative Negative Negative Negative Positive

I. API 20 E Strip test

Tests Result

ONPG POSITIVE

ADH NEGATIVE

LDC NEGATIVE

ODC NEGATIVE

CIT POSITIVE

H2S NEGATIVE

URE POSITIVE

TDA NEGATIVE

IND NEGATIVE

VP POSITIVE

GEL NEGATIVE

GLU POSITIVE

MAN POSITIVE

INO POSITIVE

SOR POSITIVE

RHA POSITIVE

SAC POSITIVE

MEL POSITIVE

AMY POSITIVE

ARA POSITIVE

OX POSITIVE

DISCUSSION

The IMViC test is combination of 4 chemical tests that are Indole, Methyl-red, Voges

Proskauer and Citrate test. 5 different cultures were used to undergo this test. For the Indole test,

the positive result changes the colour of test tube into red, as the bacteria convert tryptophan

amino acid into indole. The indole is detected using Kovac’s reagent. For culture A which is

Escherichia coli, the result should be in positive. But, the result that we got after incubated is

negative. The incorrect result obtained is maybe due to the contamination culture. Plus, they are

also possibilities that the culture is too old, means that they had been incubating for many days. In

my reading, the old culture cannot be used in Indole test because the indole is volatile and

degradable. The other bacteria culture which is bacteria A, E, H, J and Klb gave the yellowish orange

in colour. Therefore, it concluded that they did not have an enzyme tryptophanase that convert

tryptophan to indole.

BACTERIA OBSERVATION IN COLOUR FORMED

A B C D

A Light bluish-yellow

Yellow Yellow Yellow

Klb Yellow Yellow Purple Purple

MC Yellow Yellow Purple Purple

MV Yellow Yellow Purple Light bluish-purple

For the Methyl-red test, the positive test is obtained when red in colour formed in the test

tube after incubate. The bacteria A and H gave the positive result while the bacteria E, J and Klb gave

the negative result. The positive result in Methyl-red test proves that the bacteria hydrolyzed

glucose in the broth into acid. The acid produced lower the broth culture and changes the colour

into red. Meanwhile, the culture that did not produce acid condition can be seen yellow in colour.

For the Voges-Proskauer test, the positive test is obtained by the red colour appears in the

test tube. The bacteria A, E and Klb gave the positive result while the bacteria H and J gave the

negative result. The positive result due to presence of acetoin, which is an intermediate form of 2,3-

butenediol in the test tube. Plus, it proves that the bacteria contain the enzyme cytochrome oxidase.

The Voges- Proskauer test also can be used to detect a coliform.

For the Citrate utilization, the positive test is shown by the formation of blue colour. Bacteria

E, J and Klb gave the positive result while bacteria A and H gave the negative result. The purpose of

doing this test is to detect the presence of permease complex, the structure that transports citrate

into the cell. Thus, it proves that bacteria E, J and Klb have this complex.

The Oxidase test is the test to detect the presence of cyctocrome oxidase or also being

known as indophenol oxidase. In this experiment, 3 different cultures were used; A, J and K. The

positive result will occur when the bacteria that contain this enzyme reduced the Kovac’s reagent ( a

reagent that contain tetra-methly-p-phenylenediamine dihydrochloride) to become an oxidized

coloured product, which is dark blue in colour. The culture A gave the negative result, which is

colourless while culture J gave a positive result. Meanwhile, culture K, that is Micrococcus luteus

gave the false result. Theoretically, these bacteria should give the positive result because they

contain cytochrome c oxidase. But, we did not manage to get it. There might be some error during

undergo this test. First, the aseptic technique was poorly practised such as the inoculating loop was

not heat properly. Second, the culture that we obtained was totally contaminated. This is because

we had been shared the same culture with the entire group in the practical. Thus, the might be

possibilities that the culture was not pure anymore.

Instead of having biochemical tests, we were also having the dilution streaking on chocolate

and blood agar to observe the haemolysis activity on agar. They are two types of haemolyse; alpha-

haemolysis and beta-haemolysis. Alpha-haemolysis is said when the greenish or semi-clear zone

surrounding the culture. The beta-haemolysis is said when a clear zone surrounding the bacterial

colonies. Plus, this experiment is undergoing in anaerobic environment but in different way; one in

candle jar and one in anaerobic jar. They are three aspects that can be concluded in this experiment.

The first aspect is the alpha and beta haemolysis properties. From the observation, we can

conclude that bacteria J and F (in chocolate agar) are alpha haemolysis. This is because they are dark

or semi dark zone surrounded on the culture in both two types of anaerobic environment. In the left

hand, the bacteria G and F (in blood agar) are said to be the beta-haemolysis based on the clear zone

surrounding the bacteria.

The second aspect is the fastidious properties. In this practical, only bacteria F were used to

detect for this properties. The observation was the culture was fully grown on the chocolate agar.

Thus, it proves that bacteria F are the fastidious bacteria because it needs a lot consumption of

nutrient to grow.

The third aspect was the ability of two different places to create totally anaerobic condition.

The first one was a candle jar and the second was an anaerobic jar. From the observation we can

conclude that anaerobic jar was the best place to create an anaerobic condition based on the

darkness of haemolysis of same bacteria in two different places and the ability of obligate anaerobic

bacteria to grow. The candle jar only reduces the concentration of oxygen in surrounding by 16%. It

cannot totally remove the oxygen. Unlike anaerobic jar, it has GasPak that have the ability to remove

the oxygen in surrounding.

The Catalase test is the test to presence the enzyme catalase in bacteria. Positive result is

obtained when the oxygen bubbles are produced as the product of hydrogen peroxide being

converted into oxygen gas and water. 5 different cultures were tested; culture F, G, J, K and N. The

oxygen bubbles can be seen in culture F, G, J and K. Thus, it proves a positive result. The negative

result show by the culture N, prove that culture N did not have enzyme catalase.

The Coagulase test is the test the capability of bacteria to coagulate blood plasma that

resulting in white precipitation. Three different cultures were used; F, G and K. The result was all

cultures shown a positive result. This result also was false and incorrect. Morphologically, culture G

and K which are Staphylococcus epidermis and Micrococcus luteus were coagulase negative bacteria.

So, it should be negative and no precipitation formed. Therefore, the same reasons such as poor

aseptic techniques and contaminated culture were possibilities that might be happen and caused for

this false result.

The Triple Sugar Iron broth is used to determine the several of carbohydrate fermentations

plus production of hydrogen sulphide. The gas resulting from the carbohydrate metabolism also can

be detected using this broth. There are three carbohydrates being tested; lactose, glucose and

sucrose. In this practical, there were 7 different cultures tested; A, B, E, J, Klb, MC and MV.

Culture A, E and Klb have the same observation. The slant change to yellow in colour and

there were breakage at the butt of the test tube with the same colour. It proves that there are acid

and gas production. Therefore, the bacteria in these three cultures are glucose and lactose or

sucrose fermenter. The breakage of the butt of the test tubes was caused by the high pressure of gas

production that accumulates at that area.

Culture B and J also have the same observation. The slant change to reddish pink in colour

and there were no breakage at the butt of test tubes with same colour to the slant. The bacteria in

culture B and J were not fermenting any three carbohydrates in the agar. They catabolised peptone

and release ammonia, that made the butt of the test tubes became red in colour. Plus, they can

catabolise peptone in either aerobically or anaerobically.

When the bacteria MC were being cultured in TSI agar, it changes the colour to yellow with

no breakage at the butt of the test tube. It proves that MC is bacteria that ferment glucose, lactose

and/or sucrose. It produces acid condition and the butt of test tube did not break because the gas

produced did not have sufficient pressure to break the agar.

The decarboxylase test is the test for the presence of enzyme decarboxylase, an enzyme that

removes the carboxyl group from an amino acid. Every bacterium has specific decarboxylase enzyme

that only ferment or hydrolyzes specific amino acid. 4 types of different test tubes were used with 4

types of culture being tested. The test tube A is test for dextrose fermenter detection. The positive

result can be seen by the forming of yellow in colour. This is because bacteria that ferment dextrose

(a type of carbohydrates) will produce an acidic condition and turns the colour in test tube into

yellow. While the rest of three test tubes to test for the arginine, lysine and ornithine fermenter. The

positive result can be obtain by the changes of the colour of test tubes into purple colour as alkaline

condition being made from the activity of amino acids fermentation. Based on the result obtained,

we can conclude that bacteria A are not arginine, lysine and ornithine fermenter. In other word, we

can say that bacteria A do not have arginine, lysine and ornithine decarboxylase enzyme. The

bacteria A were a dextrose fermenter, which ferment dextrose and cause an acidic condition. Thus,

make the colour inside the test tube turns to yellow colour. The bacteria Klb, MC and MV have the

lysine and ornithine decarboxylase enzyme.

In string tests, 5 different culture were used; A, F, H, J and MV. From the observation only

bacteria form culture MV form a viscous solution, proved that bacteria MV is in the Vibrio group. The

viscous solution is due to the forming of string of DNA.

The API 20 E test is a multitest system of detecting and identifying the properties of bacteria.

It combines 20 different tests in one test. In this practical, bacteria from culture P were used. The

function of the test is described in the table below.

Based on the result, the bacteria P postive toward beta-galactosidase, citrate utilization,

urea hydrolysis, acetoin production, glucose fermentation/oxidation, mannitol fermentation

/oxidation, inositol fermentation/oxidation, sorbitol fermentation/oxidation, rhamnose

fermentation/oxidation, sucrose fermentation/oxidation, melibiose fermentation/oxidation,

amygdalin fermentation/oxidation and arabinose fermentation/oxidation tests.

In the left hand, the bacteria P negative toward arginine dihydrolase, lysine decarboxylase,

ornithine decarboxylase, H2S production, deaminase, indole production and gelatinase tests.

By using the Advanced Bacterial Identification Software online, the result that obtains show

that they are 4 possibilities that can be conclude. The result shown in the figure below:

Therefore, we can conclude that the bacteria in culture P might be a Klebsiella pneumonia based on

the highest accuracy in the list.

DISCUSSION

They are many biochemical tests that being invented by the scientist in order to further the

information about certain bacteria. In addition, it is also widen up the way to identify the bacteria.

Thus, it easy for us to differentiate them into respective group. The example of biochemical tests

that being applied are IMViC test, Citrate test, Oxidase test, Catalase test, Decarboxylase test,

Coagulase test, blood and choco haemolysation, Triple Sugar Iron test. To undergo all the tests, we

have to fully practise the aseptic technique in order to avoid the contamination of the culture. They

are also a system that combines several type of test in one test. The API 20 E test is a multitest

system that combines 20 different tests in one test. This system saves the culture used and easy to

handle. Thus, the identification and classification become more easier.

QUESTION

1. Discuss the theory or principle behind each test (1 to 6).

IMViC test is the combination of Indole, Methyl Red, Voges-Proskauer and Citrate utilization

tests. The purpose of Indole test is to identify the bacteria that have the presence of set enzymes

that convert typtophan to indole. The bacteria are inoculated in the test tube a medium containing

the tryptophan, a type of amino acid. The organisms that have that particular enzyme will

breakdown the tryptophan into indole. The formation of indole can be detect using Kovac’s reagent.

The Methyl-red is the test of detecting the presence of acid condition produced by the bacteria. The

bacteria are cultured in MR-VP broth with the methyl-red indicator being added. The presence of

acid causes the colour of indicator change. The Voges-Proskauer is the test to detect the presence of

enzyme cytochrome oxidase in bacteria. The bacteria are cultured in MR-VP broth with alpha

naphtol and potassium hydroxide-creatine being added. If the bacteria contain this enzyme, it will

change the original colour of the broth to pink in colour. The Citrate utilization is the test to detect

the presence of permease complex, an enzyme that transports citrate into the cell. The bacteria are

inoculated into citrate agar medium in which citrate is the sole carbon source. The blue colour will

form, resulting of the positive result of this test.

The Oxidase test is to detect the presence of cyctochrome oxidase in the certain bacteria.

Two or three drops of an oxidase test reagent, tetra-methyl-p-phenylenediamine dihydrochloride

are added to the bacteria on a piece of filter paper. The positive result of this result occur when the

reagent change it colour to blue, purple or black.

The nature of Catalase test is to identify the presence of catalase. The hydrogen peroxide is

dropped on the culture (some culture was placed on a glass slide). The formation of bubbles proves

that the catalase oxidises hydrogen peroxide to oxygen gas and water.

The TSI tests contain three types of carbohydrates of sugar; glucose, sucrose and lactose. In

addition, it also provides other additional nutrient such nitrogen, vitamins and minerals. the TSI agar

provides two condition of reaction for bacteria; the chemical reaction in aerobic condition (surface

of agar) and the chemical reaction in anaerobic condition (butt of agar). The observation of TSI can

be concluded in many ways:

Yellow colour formed :fermentation of carbohydrates that produce acidic condition and

lower the pH of agar.

Red colour formed :peptone hydrolization cause an alkaline condition, raise up the pH of

agar.

Black precipitate formed : the reduction of sodium thiosulphate to hydrogen sulphide. The

hydrogen sulphide than react with ferric ion to produce iron sulphide.

The breakage at the butt :due to high pressure of gas produced

The decarboxylase test is the test for the presence of enzyme decarboxylase, an enzyme that

removes the carboxyl group from an amino acid. Every bacterium has specific decarboxylase enzyme

that only ferment or hydrolyzes specific amino acid. The Moeller broth is a basal broth that test for

dextrose fermenter detection. The positive result can be seen by the forming of yellow in colour.

This is because bacteria that ferment dextrose (a type of carbohydrates) will produce an acidic

condition and turns the colour in test tube into yellow. In addition, the Moeller broth can be added

with different amino acid such as arginine, lysine and ornithine. The purpose of adding the three

different amino acids are to test for the arginine, lysine and ornithine fermenter. The positive result

can be obtain by the changes of the colour of test tubes into purple colour as alkaline condition

being made from the activity of amino acids fermentation.

String test is test to differentiate Vibro species from other species. Sodium deoxycholate, a

detergent that used has the ability to lyse Gram-negative organisms. When cell are lysed, DNA is

released into the suspending medium making it very viscous and able to form ‘strings of DNA’ when

touched with a loop that is raised from the surface of the liquid. The positive result achieved when

string of DNA formed.

The API 20 E strip consists of 20 microtubes containing 20 different dehydrated substrates.

The advantages of these systems are save cost and space and incubation plus provide an efficient

and reliable means of making positive identification of infectious organisms. The 20 API E is a

standardized identification system for Enterobacteriaceae and other non-fastidious. The API 20E

strip also need a period of incubation before observe the changes of colour.

2. What is the theory or principle of the GasPak reactions in an anaerobic jar?

The GasPak is a sachet that use in production of anaerobic condition to bacteria. The composition of

the GasPak sachet is sodium borohydride, sodium bicarbonate, citric acid and cobalt chloride. Each

of this plays an important role in order to create free oxygen environment. The sodium borohydride

(NaBH4) react with water vapour in jar to produce hydrogen gas.

NaBH4 + 2 H2O = NaBO2 + 4 H2↑

The citric acid reacts with sodium bicarbonate and cobalt chloride (the catalyst) to produce carbon

dioxide and hydrogen gas. The catalyst also the product of reaction due to its properties. This

reaction increases the composition of carbon dioxide and hydrogen gas.

C3H5O(COOH)3 + 2 NaHCO3 + [CoCl2] = C3H5O(COONa)3 + 3 CO2 + 3 H2 + [CoCl2]

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






Triple Sugar Iron. Retrieved 21 November, 2012, from

http://www.austincc.edu/microbugz/triple_sugar_iron_agar.php

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

compilation of the bacteriology practical report

Documents

crystal violet

differential staining

fast stain

anionic dyes

gram stain

neelsen acid

stain

bacteria