ONTENTS CONVENTIONAL METHODS AND TECHNIQUES USED IN BACTERAIL IDENTIFICATION
SUBMITTED TO: Dr. Ghazala Naseem SUBMITTED BY: Wajiha Iram: 02 Nadia Jabeen: 09 Nosheen Sharif: 28
INSTITUTE OF MYCOLOGY AND PLANT
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Topic 1- Introduction of bacteria 2- Identification of bacteria a. Conventional method i. Morphological characteraization • Size, color, margin, elevation, form, surface, opacity •
Bacterial shapes
ii. Staining techniques • Types of stains • Types of staining • Simple staining • Gram staining • Spore staining • Capsule staining • Flagella staining iii. Biochemical characterizaztion • Carbohydrate utilization • Citrate utilization • Gelatin utilization • Starch hydrolysis • Indole test • MRVP test • Triple iron & hydrogen sulphide production • Urea test • Catalase test • Oxidase test • Biochemical kit iv. Additional non- biochemical tests • Motility test References
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Introduction Bacteria are a large group of unicellular, prokaryote, microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals. Bacteria are ubiquitous in every habitat on Earth, growing in soil, acidic hot springs, radioactive waste, water, and deep in the Earth's crust, as well as in organic matter and the live bodies of plants and animals. There are typically 40 million bacterial cells in a gram of soil and a million bacterial cells in a millilitre of fresh water; in all, there are approximately five million (5×1030) bacteria on Earth, forming much of the world's biomass. Bacteria are vital in recycling nutrients, with many steps in nutrient cycles depending on these organisms, such as the fixation of nitrogen from the atmosphere and putrefaction. However, most bacteria have not been characterized, and only about half of the phyla of bacteria have species that can be grown in the laboratory.
Identification of bacteria Identification of bacteria is done by • Conventional method • Advance techniques for microbial identification
Conventional methods There are three conventional methods for bacterial identification: • Morphological characterization • Staining techniques • Biochemical characterization
1- Bacterial morphological characterization Morphological characterization of bacteria is generally carried out on the basis of colony. Following are the key steps for the morphological identification of bacterial colony.
Size For the calculation of size of bacterial colony the measurement is taken from four different sides, the average of four measurements will be the size of colony.
Color The variation in the color of bacterial colony is not actually seen as in case of fungus. The colors of bacterial colonies are mostly white, off white, yellow, and sometimes orange. The bacterial colonies also have pink color in the case of lactobacillus on blood agar.
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Form The shape of bacterial colony is irregular (ameoboidal), circular, ellipsoidal, spindle (lens), punctiform, filamentous and rhizoidal.
Elevation Elevation of bacterial colony may be flat, raised, convex, concave, umbonate(dome shape), pulvinate.
Margin The margin of bacterial colony is of six types, entire (round), undulate (wavy), filamentous (rhizoidal), lobate (having lobes), reose (serrate, dentate), curled.
Fig 1. Form, Elevation and Margin of bacterial colony
Surface The surface of bacterial colony may be either smooth or rough. The smooth colony in case of bacteria is mostly shiny. The rough colony may be coarse or granular. The coarse means finely crushed colony as compared to granular colony.
Opacity The bacterial colony may be transparent and translucent.
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Culture
Reaction
Description
Culture
Reaction
Description
Escherichia coli
This microbe forms medium sized colonies Klebsiella with a regular pneumoniae margin and convex elevation
This microbe forms slightly gummy/wet looking colonies that are circular, convex with an entire margin.
Serratia marcescens
Colonies of this microbe are red in appearance, Pseudomonas circular and fluorescens have an entire margin
Colonies are circular, convex with an entire margin
Chromobacteriu m violaceum
Colonies are circular, convex with an entire Micrococcus margin and luteus appear purple on most medium.
Colonies are punctiform, convex with an entire margin and appear yellow on most medium
Enterococcus faecalis
Colonies are punctiform, Bacillus convex with an cereus entire margin.
Colonies are large, irregular and flat with an undulate margin
Colony morphology of different bacteria
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Bacterial shapes There are three basic shapes that bacterial cells adopt. They are round, rod shaped or spiral. Round bacteria are referred to as cocci and rod shaped bacteria are known as bacilli. The term 'bacillus' meaning a rod-shaped bacterium should not be confused with the genus of bacteria known as 'Bacillus'. The shape of bacterial cells is of fundamental importance in the classification and identification of bacteria. Although bacteria are of three basic shapes, they display an astonishing variety of forms when viewed microscopically.
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Staining techniques Staining Bacteria Types of strains Basic strains, due to their positive charge will bind electrostatically to negatively charged molecules such as many polysaccharides, proteins and nucleic acids. Some commonly used basic stains are crystal violet, safranin and methylene blue. Basic strains may be used alone (a simple stain) or in combination (differential stain) depending on the experiment involved. Acids stains bind to positively charged molecules, which are much less common, meaning acidic strains are used only for special purposes.
Types of staining • Simple staining • Gram staining
1- Simple staining: When a single staining-reagent is used and all cells and their structures stain in the same manner, the procedure is caIled simple staining procedure. This procedure is of two types: positive and negative. • In positive staining, the stain (e.g., methylene blue) is basic (cationic) having positive charge and attaches to the surface of object that is negatively charged. • In negative staining, the stain (e.g., India ink, nigrosin) is acidic (anionic) having negative charge and is repelled by the object that is negatively charged, and thus fills the spaces between the objects resulting in indirect staining of the object.
Fig. 3 Simple staining of microbial cell
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Procedure Step 1. Bacteria from colonies Clean a glass slide and place a small mark slightly off center using a grease pencil. By using loop, transfer one small drop of water to the center of the slide, being careful to be close to but not overlapping the grease pencil mark. Do not transfer too much water because these drops will have to air dry. Sterilize loop and touch a single colony and transfer the bacteria to the water droplet on the slide and mix well. On mixing if see an opaque slurry of bacteria on the slide you have too many bacteria for effective staining. Bacteria from broth Clean a glass slide. Mix the broth containing the bacteria well because the bacteria may sediment to the bottom of the container. Use a sterile loop and transfer one or two droplets of bacteria to the center of a cleaned glass slide. Step 2. Drying Allow the bacterial smear to air dry. Don’t heat the sample or below it to hasten drying time because that could force bacteria into the air leading to contamination and possible infection. Step 3. Heat fixation Holding the slide by one edge, pass it slowly through a bunsen burner flame. Don’t move so slowly that the edge of the slide heat up to uncomfortable levels. This heat fixation step denatures bacterial proteins causing the cells to stick to the slide while also killing the bacteria making them safe for the following steps.
Step 4. Staining Place the stain in a staining rack and cover the smear with the stain of choice. Allow the stain to work for 30 seconds (some stains may have different staining times but this time will work well for simple stains). Remove the stain by rinsing with water from the squeeze bottle and gently blot the stain dry using bibulous paper. The slide is now ready to look at under the microscope. Because the bacteria were heat fixed, it will not be necessary to use a cover slip. 8
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Fig. 4 Preparation of smear and simple staining
Gram staining / Differential Staining Bacteria may be conveniently divided into two further groups, depending upon their ability to retain a crystal violet-iodine dye complex when cells are treated with acetone or alcohol. This reaction is referred to as the Gram reaction: named after Christian Gram, who developed the staining protocol in 1884. This reaction, however, reveals fundamental differences in the structure of bacteria. Electron microscopy shows that Gram-negative and Gram-positive bacteria have fundamentally different structures, related to the composition of the cell wall, amongst other things. Cells with many layers of peptidoglycan can retain a crystal violet-iodine complex when treated with acetone. These are called Gram-positive bacteria and appear blue-black or purple when stained using Gram's method. Gram-negative bacteria have only one or two layers of peptidoglycan and cannot retain the crystal violet-iodine complex. These need counterstaining with another dye to be seen using Gram's method. The cell wall of Gram-positive bacteria lies beyond the cell membrane and is largely made up of pepidoglycan. There may be up to 40 layers of this polymer, conferring enormous mechanical strength on the cell wall. Other polymers including teichoic and teichuronic acids also lie in the cell walls of Gram-positive bacteria. These act as surface antigens.
Fig. 5 Gram-positive bacteria
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In contrast to Gram-positive cells, the cell envelope of Gram-negative bacteria is complex. Above the cell membrane is a periplasm. This area is full of proteins including enzymes. One or two layers of peptidoglycan lie beyond the periplasm. Gram-negative bacteria are thus mechanically much weaker than Gram-positive cells. Beyond the peptidoglycan of the Gram-negative cell wall lies an outer membrane. This has protein channels - porins - through which some molecules may pass easily. The outer side of the Gram-negative outer membrane contains lipopolysaccharide.
This provides the antigenic structure of the surface of Gram-negative bacteria and also acts as endotoxin. It is this that is responsible for eliciting the symptoms of Gramnegative shock if it gains access to the bloodstream. Porins and Outer Membrane Proteins (OMPs) act as transporters through the outer membrane.
Fig. 6 Gram-nrgative bacteria A few medically important bacteria do not stain easily using conventional stains, and need to be heated to near boiling point in the chosen dye (carbol fuchsin for light microscopy: rhodamine-auramine for fluorescence microscopy) for at least five minutes. This is to allow the dye to penetrate the waxy cell walls. Having taken the stain, these bacteria resist decolourisation with both acids and alcohol, and are known as acidalcohol fast bacteria. This is a property of mycobacteria. These include Mycobacterium tuberculosis, the cause of tuberculosis; a chronic infection. Most common is pulmonary tuberculosis, affecting the lung. The kidneys may be infected in renal TB, and there is a rare form of osteomyelitis (bone infection) and meningitis caused by TB. In miliary tuberculosis, the infection is disseminated through the body. Another medically important mycobacterium is Mycobacterium leprae, the cause of leprosy; a chronic infection of the skin and nerves. Nerve damage leads to a loss of sensation, and ultimately to paralysis. This can lead to tissue damage that can lead to the loss of fingers and toes.
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Procedure Step 1. Bacteria from colonies Sterilize loop and touch a single colony and transfer the bacteria to the water droplet on the slide and mix well. Bacteria from broth Clean a glass slide. Mix the broth containing the bacteria well because the bacteria may sediment to the bottom of the container. Use a sterile loop and transfer one or two droplets of bacteria to the center of a cleaned glass slide. Step 2. Drying Allow the bacterial smear to air dry. Don’t heat the sample or below it to hasten drying time because that could force bacteria into the air leading to contamination and possible infection. Step 3. Heat fixation Holding the slide by one edge, pass it slowly through a bunsen burner flame. Don’t move so slowly that the edge of the slide heat up to uncomfortable levels. This heat fixation step denatures bacterial proteins causing the cells to stick to the slide while also killing the bacteria making them safe for the following steps.
Step 4. Primary stain Cover the smear with crystal violet and incubate for 30 seconds. Rinse the dye off with distilled water from squeeze bottle. Step 5. Mordant Cover the smear with gram iodine. After 20 seconds, rinse the slide with distilled water. Step 6. Decolorization Rinse the stain with 95% ethanol. This step must be done very carefully. Hold the slide at 45o angle over the staining rack and rinse with ethanol one drop at a time. Watch the ethanol as it runs off the slide looking for blue color. Stop dropping ethanol as soon as no more color is releases and rinse the slide immediately with water. A few drops of ethanol too many and the gram positive bacteria will also lose their crystal violet. Step 7. Counterstain Cover the bacteria with safranin for 30 seconds. Rinse with distilled water and blot the slide dry with bibulous paper. Observed under microscope.
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Spore staining Bacterial spores (or endospores) are the toughest forms of life known. They are so resistant to destruction that some scientists have proposed that life arrived on earth when bacterial spores drifting through space fell to earth. Only G+ cells form spores, specifically members of the genera Bacillus and Clostridium. Spores are formed by bacteria to survive during period of deprivation, such as the loss of a food or water supply. When a spore-forming-bacterium (SFB) senses that tough times are coming a series of complex events are triggered that lead to the formation of a spore. Because a thick, tough covering protects the spore, it is difficult to stain. Usually following procedure is followed for spore staining.
Procedure 1. Using an inoculation loop, prepare a smear of microorganism; allow the smear to air dry. 2. Heat fix the smear by passing it through the bunsen burner flame a few times. 3. Flood the smear with malachite green. 4. Place the slide on a tall staining rack. Pass the burner flame under the slide until the stain steams; continue for 5 minutes, replenishing the stain as needed. Do not allow the stain to boil or completely evaporate. 5. Remove the samples from the heat source and allow the slides to cool. 6. Rinse the slides with water. 7. Flood the sample with safranin for 30-60 seconds. 8. Rinse the slides with water. 9. Blot dry with bibulous paper. Examine under the microscope.
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Fig. 7 Bacillus subtilis, spore stain.
Capsule staining Some bacteria have characteristic surface structures: capsules or flagella and internal components: endospores, which may have taxonomic value for their identification. When it is necessary to demonstrate whether or not a particular organism possesses a capsule, is flagellated, or forms endospores, special staining techniques must be used. Many bacteria possess a capsule, but it is often not visible. A few pathogenic species, however, such as Streptococcus pneumonia, Klebsiella pneumonia, and Clostridium perfringens, have welldeveloped capsules that contribute to virulence by protecting the organisms from host defense mechanisms, particularly phagocytosis. Capsular materials often are antigenic, providing the bacterial cell with specific immunologic properties by which they may be identified. Capsules can be visualized microscopically by using a simple, nonspecific negative staining technique. The negative staining procedure is less distorting to cells. Stains like Nigrosin are negatively charged (-) and are repelled by the negative change (-) of the bacteria. This prevents them from entering the cell and staining the cytoplasm. This technique produces a stained background which surrounds the unstained microbe.
Figure 8. Capsule staining of bacteria
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After staining the cells with Nigrosin, the slides are dried, and the slide preparation is stained with safranin, a dye that does penetrate the cells and stains them. When viewed under the microscope after this treatment, the bacteria are pink, while their capusles appear as clear, unstained zones surround them, their outlines demarcated against the black backgound proved by Nigrosin.
Flagella Staining Bacterial flagella are composed of protein subunits called flagellin and are distinct in structure from flagella found on eukaryotic cells. Flagella are anchored to the bacterial cytoplasmic membrane and cell wall by basal bodies, and are assembled via flagellin subunits traveling through the basal bodies, then through the center of the flagellum itself, and are added to the distal end of the appendage. The intrinsic structure of the flagellum is helical, making propulsion possible. Flagella are of variable lengths that can extend several times the length of the bacterium itself, with widths generally between 10 and 50 nm. Due to their narrow width the best direct method of observing bacterial flagella is by the electron microscope, because the normal limit of resolution of light microscopy is ~200 nm. For those who do not have access to electron microscopy, bacterial flagella can be observed via the light microscope in combination with stains. All flagella stains use mordants, like tannic acid and potassium alum, to coat and thus thicken the flagellum in order to be within the limits of size observable by light microscopy. The Leifson flagella stain method uses tannic acid. For the Leifson flagella stain, tannic acid and the dye form a colloidal precipitate that when absorbed by the flagellum causes it to increase in diameter and become colorized, thus amenable to viewing by light microscopy. The tannic acid-dye complex is more soluble in alcohol than water, and also more soluble with decreased pH. The alcohol concentration in the Leifson solution is sufficient to maintain solubility of the components. When the prepared sample is stained, the alcohol evaporates faster than the water, and the concentration of the tannic acid and dye increases to cause precipitation, leading to staining of the flagella (Silflow & Lefebvre, 2001). Salt concentration also affects the staining, presumably by altering charge of the tannic acid-dye complex and the flagellum itself. The flagella stain is finicky because many variables affect the outcome: • •
Age of the bacterial culture Thickness of the culture on the microscope slide
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• • • • • •
Age of the staining solutions, pH Temperature Alcohol concentration Dye concentration Heat
Procedure 1.
Take a prepared slide and using a wax pencil draw a rectangle around the dried sample. Place slide on staining rack.
2. Flood Leifson dye solution on the slide within the confines of the wax lines. Incubate at room temperature for 7 to 15 minutes. The best time for a particular preparation will require trial and error.
3.
As soon as a golden film develops on the dye surface and a precipitate appears throughout the sample, as determined by illumination under the slide, remove the stain by floating off the film with gently flowing tap water. Air dry.
4. View using oil immersion, at 1,000x magnification, by bright-field microscopy. Bacterial bodies and flagella will stain red.
Fig. 9 Staining of Vibrio choleraeby using Liefson's flagellar stain method.
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Biochemical Tests for Identifying Unknowns Carbohydrate Utilization Bacteria produce acidic products when they ferment certain carbohydrates. The carbohydrate utilization tests are designed to detect the change in pH which would occur if fermentation of the given carbohydrate occurred. Acids lower the pH of the medium which will cause the pH indicator (phenol red) to turn yellow. If the bacteria do not ferment the carbohydrate then the media remains red. If gas is produced as a by product of fermentation, then the Durham tube will have a bubble in it. Following carbohydrate tests were performed: • • •
Glucose (Dextrose) test Lactose test Sucrose test
A
B
C
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Fig 10. Fermentation results from left to right; A: shows less acid formation than far right tube, but gas is still made. B: no carbohydrate utilization to produce acid or gas. C: shows acid was produced as evidenced by the yellow color, and gas was made
Citrate Utilization Tests for the ability of bacteria to convert citrate (an intermediate of the Kreb’s cycle) into oxaloacetate (another intermediate of the Kreb’s cycle). In this media, citrate is the only carbon source available to the bacteria. If it can not use citrate then it will not grow. If it can use citrate, then the bacteria will grow and the media will turn a bright blue as a result of an increase in the pH of the media. To inoculate this slant, use the transfer loop.
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Gelatin Utilization This media is used to test if bacteria can digest the protein gelatin. To digest gelatin, the bacteria must make an enzyme called gelatinase. To inoculate this media, use a transfer needle to stab the gelatin. After incubating the inoculated media for at least 48 hrs, transfer the tube into a refrigerator. The tube should be completely chilled prior to observation. If the media is solid after refrigeration then the test is negative (the bacteria did not digest gelatin). If the media is liquefied even after refrigeration, then the test result is positive…the bacteria is able to digest gelatin.
A
B
Fig.12. A: shows 'Serratia marcescens' which is positive for gelatinase production, as evidenced by the liquidation of the media. B: 'Salmonella typhimurium' shows negative results for gelatinase production, as evidenced by the solidity of the media.
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Starch hydrolysis This test is used to detect the enzyme amylase, which breaks down starch. After incubation the plate is treated with Gram’s iodine. If starch has been hydrolyzed (broken down) then there is a reddish color or a clear zone around the bacterial growth; if it has not been hydrolyzed then there is a black/blue area indicating the presence of starch. Simply use inoculating loop to spread bacteria onto plate surface. After the bacteria have grown, add a few drops of Gram’s iodine to the plate and look for the color immediately after adding the iodine.
Fig 13. Starch hydrolysis test in E.coli
Indole production This test is done to determine if bacteria can breakdown the amino acid tryptophan into indole. SIM media or TSB (tryptic soy broth) is inoculated using a transfer needle. After incubating the bacteria for at least 48 hours, Kovac’s reagent is 20
added to the media to detect if indole has been made by the bacteria. The development of a red/pink layer on top of the media is a positive result (the bacteria can breakdown tryptophan to form indole). Failure to see a red layer is a negative result (indole was not formed from tryptophan).
A
B
FIG 14. A: showing red ring formation is positive for indole production. B: shows a negative result.
MRVP (methyl red-Vogues Proskauer) This test is used to determine two things. The MR portion (methyl red) is used to determine if glucose can be converted to acidic products like lactate, acetate, and formate. The VP portion is used to determine if glucose can be converted to acetoinThese tests are performed by inoculating a single tube of MRVP media with a transfer loop and then allowing the culture to grow for 3-5 days. After the culture is grown, about half of the culture is transferred to a clean tube. One tube of culture will be used to conduct the MR test, the second tube serves as the VP test.
A. MR (methyl red) test
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Methyl red is added to the MR tube. A red color indicates a positive result (glucose can be converted into acidic end products such as lactate, acetate, and formate. A yellow color indicates a negative result, glucose is converted into neutral end products.
B.
VP (Vogues Proskauer) test First alpha-napthol (also called Barritt’s reagent A) and then potassium hydroxide (also called Barritt’s reagent B) are added to the VP tube. The culture should be allowed to sit for about 15 minutes for color development to occur. If acetoin was produced then the culture turns a red color (positive result); if acetoin was not produced then the culture appears yellowish to copper in color (a negative result).
Triple sugar Iron (TSI) - & Hydrogen sulfide production (H2S) Looks at fermentation of glucose, lactose, and sucrose and checks if hydrogen sulfide is produced in the process. Basically a pH indicator will change the color of the media in response to fermentation, where that color change occurs in the tube will indicate what sugar or sugars were fermented. The presence of a black color indicates that H2S was produced. In this media, H2S reacts with the ferrous sulfate in the media to make ferrous sulfide, which is black. To inoculate, use a needle to stab agar and then uses a loop to streak the top slated region. In addition to TSI media, SIM media can be used to determine if H2S is produced. A black color in the SIM medium following inoculation and incubation indicates that H2S is made by the bacteria. SLANT COLOR RED YELLOW
Interpretation does not ferment either lactose or sucrose ferments lactose and/or sucrose
BUTT COLOR/CONDITION
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RED YELLOW
no fermentation of glucose some fermentation of glucose has occurred, acid has been produced Seen as cracks in the agar, bubbles, or the entire slant may be pushed out of the tube. H2S has been produced
GAS FORMED BLACK
A
B
C
D
E
F
G
H
FIG. 17. Triple sugar Iron (TSI) - & Hydrogen sulfide production (H2S). A: Uninoculated control; B: Red slant and red butt, no black color i.e., no fermentation of glucose, sucrose or lactose. No Hydrogen sulfide produced; C: Red slant and black butt i.e., no lactose or sucrose fermentation, H2S has been produced; D: Red slant with yellow butt i.e., no
lactose or sucrose fermentation, lactose is fermented, no H2S has been produced ; E: Yellow slant, yellow butt and black coloration i.e., Lactose, sucrose and glucose fermented, and H2S has been produced; F & G: Yellow slant, yellow butt and lifting
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and/or cracking of media, no black coloration i.e., Lactose, sucrose and glucose fermented, H2S has not been produced but gas has been produced; H: Yellow slant, yellow butt and no lifting and/or cracking of media, no black coloration i.e., Lactose, sucrose and glucose fermented, H2S has not been produced nor has gas been produced.
Urea test This test is used to detect the enzyme urease, which breaks down urea into ammonia. Ammonia is a base and thus will raise the pH of the media if it is present. This change in pH is indicated by a pH indicator called phenol red which is present in the media. A color change from yellow to bright pinkish-red is positive; lack of color change is a negative result. Inoculate the liquid media with a transfer loop. A
B
C
Fig.19 urease test; A: shows positive reaction; B: shows negative reaction; C: shows uninoculated control.
Catalase Test This test is can be used to detect the enzyme catalase. This enzyme is responsible for protecting bacteria from hydrogen peroxide (H2O2) accumulation, which can occur during aerobic metabolism. If hydrogen peroxide accumulates, it becomes toxic to the organism. Catalase breaks H2O2 down into water and O2. To perform the catalase test simply smear a small amount of the test organism onto the lid of a Petri plate/culture dish. Then add a drop of hydrogen peroxide to the smear. If bubbles become visible (these would be the O2 bubbling up) then the test is positive and you can conclude that the organism makes catalase. A lack of bubbles indicates the absence of catalase. *Note, most aerobic organism make catalase.
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Fig 21. Bubbling upon the addition of hydrogen peroxide is indicative of the presence of catalase for this organism.
Oxidase test To perform this test simply swab some of your test culture into one of the boxes on an oxidase dry slide. If a color change to purple or blue is evident at 30 seconds-1 minute then the result is positive. It is important that the test is read by one minute to ensure accurate results (avoid false negatives and false positives). This laboratory test is based on detecting the production of the enzyme cytochrome oxidase by Gram negative bacteria. It is a hallmark test for the Neiserria. It is also used to discriminate between aerobic Gram-negative organisms like Pseudomonas aeruginosa and other Enterobacteriaciae.
Biochemical kit
• • •
Because for the identification of a bacteria at specie level 25-50 biochemical tests are applied so it is laborious and compensated by the use of biochemical kits. Biochemical kits comprises of; wells supplied with chemicals saline water for bacterial suspension chemicals for reaction
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Procedure The chemicals present in the contain sugars and antibiotics solutions. While the gel, production of nitrate and nitrites, and production of CO2 . This is done with saline water contains bacterial suspension made with help of spectrophotometer. Then this suspension was poured in the wells and checks the hydrolysis the help of different solutions. The most common used solutions are gel, nitrate solution and VP solution. All the data from these reactions are checked with help of a catalog which contains different positive and negative reaction .by this catalog we get a cod which is universal, this cod will tell us the species of bacteria. All these methods are conventional methods. Now we use the modern techniques for the identification of bacteria.
API-20 strip test
O A L O C H U T I G G M I S R S M A A N V culture D D D I 2 R D N E L A N O H A E M R identification P P no. H C C T S E A D L U N O R A C L Y A G 8101
+ – + + – – – – + – – + + – + + + + – +
Escherichia coli
5B
+ – – – + – – – – + – + + + + + – – + +
Enterobacter agglomerans
8P44
– – + + – + – – + – – + + – – – + – – +
Edwardsiella hoshinae
The types of biochemical tests we have explored here can be miniaturized. The API-20 strip is one example of this type of test. Twenty tests are performed on this strip by a simple procedure, saving time and money.
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• • • •
The first test is for the presence of the enzyme β-galatosidase, an enzyme involved in lactose catabolism. The next three reactions (in order, arginine, lysine and ornithine) test for amino acid decarboxylation. Decarboxylation is shown by an alkaline reaction (red color of the particular pH indicator used). Hydrogen sulfide production (H2S) and gelatin hydrolysis (GEL) result in a black color throughout the tube. A positive reaction for tryptophan deaminase (TDA) gives a deep brown color with the addition of ferric chloride. Positive results for this test correlate with positive phenylalanine and lysine deaminase reactions which are characteristic of Proteus, Morganella and Providencia. The last nine tests are for carbohydrate fermentation. The carbohydrates tests are (glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin and arabinose. Fermentation is shown by an acid reaction (yellow color of indicator).
Additional Non-Biochemical Tests Motility test The motility test is not a biochemical test since we are not looking at metabolic properties of the bacteria. Rather, this test can be used to check for the ability of bacteria to migrate away from a line of inoculation thanks to physical features like flagella. To perform this test, the bacterial sample is inoculated into SIM or motility media using a needle. Simply stab the media in as straight a line as possible and withdraw the needle very carefully to avoid destroying the straight line. After incubating the sample for 24-48 hours observations can be made. Check to see if the bacteria have migrated away from the original line of inoculation. If migration away from the line of inoculation is evident then you can conclude that the test organism is motile (positive test). Lack of migration away from the line of inoculation indicates a lack of motility (negative test result).
A
B
Fig. 23 A: Shows result for a non-motile bacterium; B: shows result for a motile organism.
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Identifying of microorganisms through these types of biochemical tests has been a routine practice for many decades and the tests necessary to identify certain species of groups of related species have become standardized. To make these test more convenient, manufacturers have developed miniaturized versions that decrease the material cost and make inoculation of the tests much more convenient. However, the tests still need to be incubated to the prescribed period of time before being read, usually 24-48 hours.
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