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
H -Salmonella typhi
B -Bacillus subtilis
I -Streptomyces griseus
C -Bacillus megaterium
J -Pseudomonas sp.
D -Bacillus cereus
K-Micrococcus luteus
E -Serratia sp.
L -Nocardia sp.
F -Staphylococcus aureus
M-Vibrio sp.
G -Staphylococcus epidermidis
RESULT A-COLONY MORPHOLOGY Bacteria A
Size (mm) 3
B C D
1 6 10
E
2
F G H
3 3 2-3 mm
I J K
1-2mm 1mm 1-2mm <1 mm
L M
3
Colour Creamy white White Cream Creamy White Reddish pink Cream Cream Grey
Form/texture Margin Circular Entire
Elevation Convex
Transparency Translucent
Circular Circular Rhizoid
Lobate Undulate Undulate
Raised Flat Flat
Translucent Translucent Translucent
Circular
Entire
Umbonate
Opaque
Circular Circular Round
Entire Undulate Entire
Raised Raised Raised
Translucent Opaque Translucent
White Creamy Creamy Yellowishorange Green
Circular Circular Circular Circular
Filamentous Entire entire Entire
Umbonate Umbonate Convex Raised
Opaque Opaque Opaque Translucent
Irregular
Entire
Convex
Translucent
B-GRAM STAINING Bacteria A B F G I M K
Gram-positive/negative Gram-negative Gram-positive Gram-positive Gram-positive Gram-positive Gram-negative Gram-positive
C-SPORE STAINING Bacteria A B C D I L J K
Shape ENTERIC ROD ROD ROD COCCI BRANCHED COCCI COMMA/ROD TETRACOCCI
D- ACID- FAST STAINING Spore staining NEGATIVE NEGATIVE POSITIVE POSITIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE
Bacteria B I L
Acid-Fast Staining NEGATIVE NEGATIVE POSITIVE
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.
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 Grampositive. 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, heatresistant 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 Gramstain 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/3136colony-morphology-protocol Marise A. Hussey, A. Z. (2008). Acid-Fast Stain Protocols Retrieved 15 November, 2012, from http://www.microbelibrary.org/component/resource/laboratorytest/2870-acid-fast-stain-protocols Marise A. Hussey, A. Z. (2007). Endospore Stain Protocol, from http://www.microbelibrary.org/library/laboratory-test/3112-endospore-stainprotocol 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 CFU
10-1
10-2
10-3
10-4
10-5
10-6
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 CFU
10-1
10-2
10-3
10-4
10-5
10-6
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 Red
Single colony produced 11
Size 3mm
Morphology Margin Elevation Appearance 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 Colonies formed on a surface of agar media (surface/subsurface colonies). (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 45 C 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. o
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. 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 TEST TUBE
DILUTION FACTOR 1 2 3
Number of tubes with positive result
10-4
10-5
10-6
+ + + 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
10-3 10-4 10-5
Colony-forming Unit (CFU) on plate TNTC 58 1
Colour of colony Pink Pink 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 The coliform formed on the solid agar and can undergo a direct bacteria count. 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 The MFT did not collect gas. formed. 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 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. 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 https://docs.google.com/a/siswa.um.edu.my/viewer?a=v&q=cache:DGEWvKj DVDkJ:www.mastgrp.com/Dehydrated%2520culture%2520media/InfoSheet/D M/DM150%2520MacConkey%2520Broth.pdf+durham+tubes+with+macconke y+broth&hl=en&gl=my&pid=bl&srcid=ADGEESgklBwtvYxjDkya56zwWfBN3Pr 7K2Pfv1WMflCdrGtT1wkHoS9Y1ivyOzvuTEuUxuFMUgWN7yjLuc4PXDZ218fSVZjGZ3bbhYoHaO2sgeQ YoCkzOREeoYxoWGdkT5muD1Fs4sF&sig=AHIEtbSnOQBeOb2WEu1i4Rg817SsoSbbg The hemocytometer (counting chamber). (2010). Retrieved 19 November, 2012, from http://www.microbehunter.com/2010/06/27/the-hemocytometer-countingchamber/ 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: E: F: G: H: I:
Escherichia coli Serratia sp. Staphylococcus aureus Staph. epidermidis Salmonella typhi Streptomyces griseus
J: K: N: Klb: MC: MV:
Pseudomonas sp. Micrococcus luteus Streptococcus faecalis Klebsiella sp. Vibrio cholerae Vibrio parahaemolyticus
RESULT: A. IMViC Test BACTERIA
INDOLE TEST
A E H J Klb
Negative Negative Negative Negative Negative
METHYL-RED TEST Positive Negative Positive Negative Negative
VOGES-PROSKAUER TEST Positive Positive Negative Negative Positive
CITRATE TEST Negative Positive Negative Positive Positive
B. Oxidase Test
C. Catalase Test
BACTERIA A J K
RESULT Negative Positive Negative
BACTERIA F G J K N
RESULT Positive Positive Positive Positive Negative
D. Haemolysis of blood and chocolate agar in two different anaerobic environment OBSERVATION BACTERIA
CANDLE LIGHT JAR BLOOD AGAR CHOCOLATE AGAR
ANAEROBIC JAR BLOOD AGAR CHOCOLATE AGAR
F
Clear zone
Semi clear zone
Semi clear zone
G
-
-
J
Dark, semi clear zone
Greenish, semi clear zone -
Dark, semi clear zone Clear zone
Dark, semi clear zone
E. Coagulase Test BACTERIA
RESULT
F G K
Positive Positive Positive
F. Triple Sugar Iron Test BACTERIA
OBSERVATION Slant condition
A B E J Klb MC MV
Yellow in colour Reddish pink in colour Yellow in colour Reddish in colour Yellow in colour Yellow in colour No change observed.
Butt condition Breakage with yellow in colour No breakage with reddish pink in colour Breakage with yellow in colour No breakage with reddish in colour Breakage with yellow in colour No breakage with yellow in colour No change observed.
G. Decarboxylase test BACTERIA
OBSERVATION IN COLOUR FORMED A
B
D
C
A
Light bluishyellow
Yellow
Yellow
Yellow
Klb
Yellow
Yellow
Purple
Purple
MC
Yellow
Yellow
Purple
Purple
MV
Yellow
Yellow
Purple
Light bluishpurple
H. String test BACTERIA
A
F
H
J
MV
STRING FORMATION
Negative
Negative
Negative
Negative
Positive
I. API 20 E Strip test Tests ONPG ADH LDC ODC CIT H2S URE TDA IND
Result POSITIVE NEGATIVE NEGATIVE NEGATIVE POSITIVE NEGATIVE POSITIVE NEGATIVE NEGATIVE
VP GEL GLU MAN INO SOR RHA SAC MEL AMY ARA OX
POSITIVE NEGATIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE 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.
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 2,3butenediol 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; alphahaemolysis 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
Red colour formed
Black precipitate formed
The breakage at the butt
:fermentation of carbohydrates that produce acidic condition and lower the pH of agar. :peptone hydrolization cause an alkaline condition, raise up the pH of agar. : the reduction of sodium thiosulphate to hydrogen sulphide. The hydrogen sulphide than react with ferric ion to produce iron sulphide. :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 αNaphthylamine, 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 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. 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 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/3229oxidase-test-protocol Katz, D. S. (2010). Coagulase Test Protocol Retrieved 21 November, 2012, from http://www.microbelibrary.org/library/laboratory-test/3220-coagulase-testprotocol 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/c holera/pdf/laboratory-methods-for-the-diagnosis-of-vibrio-cholerae-chapter6.pdf+vibro+cholare+on+dextrose+decarboxylase&hl=en&gl=my&pid=bl&srci d=ADGEESiZqRNj7Gly8No5Fd0SnyqVl_U_qcXuS27_QkBARVjwmqqXRbPc QcQ35wJzr8jJLio4it0Gtsce9m_0kkdhYBmO9gE1HeiMni3wJ5_2b_aY2jFqCt2H0vlpkoKlC0AJCA05mCd&sig=AHIEtbQAwIkX hQzVwLNtO0woB9C3fyQaDQ
