Bacterial Colonial Characteristics

Bacterial Colonial Characteristics ♣ Appearance in petri dish. ♣ Appearance in test tube. ♣ Affecting factor on growth ♣Culturing of anaerobic bacteria ♣features growth on media of bacterial * Colony morphology in petri dish • A colony is a large number of bacterial cells on solid medium, which is visible to the naked eye • as a separate unit. • Colony is derived from one bacterial cell. Different species of bacteria can produce very different colonies. • In the identification of bacteria and fungi much Focus is placed on how the organism grows in or on media. • Appearance of bacterial colony on culture medium is * Bacterial Colonial Features on Petri dish • Shape – Circular, irregular, rhizoid or filamentous. • Size –can be categorised as large (>1 mm), medium (=1 mm), small (<1 mm) and pinpoint (<0.5 mm). • Surface Appearance – Can be smooth, rough/granular, dull, shiny or wrinkled. • Texture or consistency – Brittle/friable (dry, breaks apart), firm, butyrous (buttery) and mucoid (sticky, mucus-like). • Elevation – Colony may be raised, flat, convex, * Bacterial Colonial Features on Petri dish • Edge – Entire, lobate, crenated, undulate or ciliate. • Colour – Pigments are produced by certain bacterial species. Prodigiosin by Serratia marcescens. Pyoverdin and pyocyanin gives Pseudomonas aeruginosa a greenish sheen. • Opacity – Transparent, translucent or opaque. • Haemolysis – Haemolysin produced by some bacteria leads to haemolysis around the colony. Based on that, the bacterial colony can be: • Alpha – Partial destruction of blood cells. • Beta – Complete destruction of blood cells. • 1. Form – The form refers to the shape of the colony. 2. Size - The diameter of a representative colony may be measured. Tiny colonies are referred to as (pin-point) Punctiform. 3. surface- smooth, shiny, rough, dull, wrinkled. 4. Texture – Several terms that may be appropriate for describing the texture or consistency of bacterial growth are: dry, moist, mucoid, hard, viscous, butyrous (buttery). Granular colony of mycobacterium tuberculosis mucoid colonies of Klebsiella pneumoniae are indication of the bacterium’s heavy capsule. 5. Color – It is important to describe the color or pigment of the colony. such as: opaque, dull, translucent, iridescent (rainbow-like). 6. Elevation – This describes the “side view” of a colony. These are the most common. 7. Margin/Edge – The margin or edge of a colony (or any growth) may be an important characteristic in identifying an organisms. entire filifor m undulate Bacillus subtilis lobate colony  Broth :growth occurs throughout the container and can then present a dispersed, cloudy, or flaky appearance. appearance in Semisolid media(slant) the medium is stabbed carefully in the center with an inoculating needle and later observed for the pattern of growth around the stab line. Tubes containing pure cultures of Escherichia coli (white), Micrococcus luteus (yellow), and Serratia marcescens (red). Morphological Tests for Identification 1.Gram test 2.Ziehl Neelson test 3.Negative Staining 4.Flagella staining test 5.Endospore test 6.Capsule test 7.Lactophenol cotton-blue stain (for Fungi/Moulds) Gram Stain Principle • Hans Christian Gram discovery in the late 19th century • Bacteria stain either Gram positive or Gram negative • Exceptions include: • • • • Chlamydia Mycoplasma Ureaplasma Spirochetes 3 Gram Positive Organisms Gram Positive • Gram positive organisms have thickly layered peptidoglycan cell walls • Gram positive organisms resist decolorization by acetone or alcohol 4 Gram Negative Organisms Gram Negative • Gram negative organisms have thin peptidoglycan cell walls surrounded by an additional lipopolysaccharide and protein outer membrane • organisms are Gram negative readily decolorized with acetone or 5 alcohol • Assess the quality of a specimen • Presence of cellular material • Accept and reject sputum • Provide rapid presumptive diagnosis of infectious agents • Classify bacteria by form, size and cellular morphology • Determine presence of yeast and hyphae • Determine course of treatment • Direct the bench work-up of a specimen 6 • • • • • • • Heat fix or methanol fix the air dried slide Flood the slide with Crystal Violet Stain for 15-30 seconds Flood slide with Gram’s Iodine Stain for 15-30 seconds Rinse slide gently with tap water Decolorize the slide until the run off is clear by allowing the decolorizer to flow over the slide held at an angle. (Time is subject to the thickness of the smear and type of decolorizer being used.) 1 0 Gram Stain Procedure (continued) • Rinse slide gently with tap water • Flood slide with Safranin (counterstain) • Stain slide for 15-30 seconds • Rinse slide gently with tap water • Drain excess water • Air dry slide in an upright position or gently blot with bibulous paper. 1 1 What’s Happening In the Gram Stain Process 1 2 What’s Happening In the Gram Stain Process 1 2 Trouble - shooting Considerations • Smear preparation • Thickness of smear • Concentration of organisms • Antibiotic therapy • Artifacts • Experience • Smear fixing • Excessive heat fixing • Inadequate fixing • Smear 1 4 Ziehl-Neelsen Staining: Principle, Procedure & Results • The Ziehl-Neelsen staining technique is a differential staining technique • initially developed by Ziehl and modified later by Neelsen, hence the name ZiehlNeelsen stain. • Neelsen used carbol-fuschin from Ziehl’s experiment, with heat and added a decolorizing agent using acid-alcohol and a counterstain using methylene blue dye, thus developing the Ziehl-Neelsen Technique of staining. • The use of acid-alcohol in the technique earned it the name Acid-Fast Stain and the application of heat in the technique gives it the name the hot method of Acid-Fast Ziehl-Neelsen Staining: Principle, Procedure & •Results This technique is used on microorganisms that are not easily stained by basic stains such as Negative staining or Gram staining. One of the most complex microorganisms that require harsh treatment of the ZiehlNeelsen compounds is the Mycobacterium spp. •Mycobacterium, Actinomycetes, Norcadia, Isospora, Cryptosporidium, and some fungi contain a thick cell wall made up of lipoidal complexes known as mycolic acid. •Mycolic acid is difficult to stain and therefore simple stains like gram staining can not penetrate the thick cell wall of these organisms. •They require harsher treatments to allow stain penetration for identification and examination and hence the use of the Ziehl-Neelsen or the hot method of Acid-fast stain. Objectives of the Ziehl-Neelsen Staining Ziehl-Neelsen Staining: Principle •The Ziehl-Neelsen stain uses basic fuchsin and phenol compounds to stain the cell wall of Mycobacterium species. •Mycobacterium does not bind readily to simple stains and therefore the use of heat along with carbol-fuschin and phenol allows penetration through the bacterial cell wall for visualization. •Mycobacterium cell wall contains high lipid content made up of mycolic acid on its cell wall making it waxy, hydrophobic, and impermeable.. •Use of Carbol-fuschin which is basic strongly binds to the negative components of the bacteria which include the mycolic acid and the lipid cell wall. addition of acid alcohol along with the application of heat forms a strong complex that can not be easily washed off with solvents. •The acid-fast bacilli take up the red color of the primary dye, carbol-fuschin. •While non-acid-fast bacteria easily decolorize on the Ziehl-Neelsen Staining: Procedure Reagents used in the Ziehl-Neelsen Stain 1.Carbol-Fuschin (Primary dye) 2.20% sulphuric acid or acid-alcohol (Decolorizer) 3.Methylene Blue dye (counterstain) or malachite green Procedure 4.On a clean sterile microscopic slide, make the smear of the sample culture and heat fix the smear over blue heat. 5.Over the smear, pour and flood the smear with carbol fuschin and heat gently until it produces fumes. 6.Allow it to stand for 5 minutes and wash it off with gently flowing tap water. 7.Add 20% sulphuric acid and leave it for 1-2 minutes. Repeat this step until the smear appears pink in color. 8.Wash off the acid with water. 9.Flood the smear with methylene blue dye and leave it for 2-3 minutes and wash with water. 10.Air dry and examine the stain under the oil immersion lens. Ziehl-Neelsen Staining: Results •Acid-fast bacteria retain the primary dye, carbol-fuschin, and stain pink. •Non-acid fat bacteria take up the methylene blue dye and appear blue. Negative Staining- Principle, Procedure & Result Interpretation Principle of Negative Staining • Negative staining requires the use of an acidic stain such as India ink or nigrosin. • The acidic stain, with its negatively charged chromogen, will not penetrate the cells because of the negative charge on the surface of bacteria. Therefore, the unstained cells are easily discernible against the colored background. • The practical application of negative staining is twofold. • • First, since heat fixation is not required and the cells are not subjected to the distorting effects of chemicals and heat, their natural size and shape can be seen. Second, it is possible to observe bacteria that are difficult to stain, such as some spirilla. • Because heat fixation is not done during the staining process, keep in mind that the organisms are not killed and slides should be handled with care. Negative Staining- Principle, Procedure & Result Interpretation Procedure of Negative Staining 1.Place a small drop of nigrosin close to one end of a clean slide. 2.Using aseptic technique, place a loopful of inoculum from the bacterial culture in the drop of nigrosin and mix. 3.Place a slide against the drop of suspended organisms at a 45° angle and allow the drop to spread along the edge of the applied slide. 4.Push the slide away from the drop of suspended organisms to form a thin smear. Air-dry. Flagella Staining- Principle, Procedure & Result Interpretation This is used to visualize the presence and arrangement of flagella for the presumptive identification of motile bacterial species. Principle Flagella are too thin to be visualized using a bright field microscope with ordinary stains, such as the Gram stain, or a simple stain. A wet mount technique is used for staining bacterial flagella, and it is simple and useful when the number and arrangement of flagella are critical to the identification of species of motile bacteria. The staining procedures require the use of a mordant so that the stain adheres in layers to the flagella, allowing visualization. Flagella Staining- Principle, Procedure & Result Interpretation Procedure 1.Grow the organism to be stained appropriately. 2.Add a small drop of water to a microscope slide. 3.Dip a sterile inoculating loop into sterile water. 4.Touch the loopful of water to the colony margin briefly (this allows motile cells to swim into the droplet of water). 5.Touch the loopful of motile cells to the drop of water on the slide. Note: Agitating the loop in the droplet of water on the slide causes the flagella to shear off the cell. 6.Cover the faintly turbid drop of water on the slide with a coverslip. A proper wet mount has barely enough liquid to fill the space under a coverslip. Small air spaces around the edge are preferable. 7.Examine the slide immediately under 40× to 50× for motile cells. If motile cells are not seen, do not proceed with the stain. Flagella Staining- Principle, Procedure & Result Interpretation Procedure 1.Gently apply 2 drops of RYU flagella stain (Remel, Lenexa, Kansas) to the edge of the coverslip. The stain will flow by capillary action and mix with the cell suspension. Small air pockets around the edge of the wet mount are useful in aiding the capillary action. 2.After 5 to 10 minutes at room temperature, examine the cells for flagella. 3.Cells with flagella may be observed at 100× (oil) in the zone of optimum stain concentration, about halfway from the edge of the coverslip to the center of the mount. 4.Focusing the microscope on the cells attached to the coverslip rather than on the cells attached to the slide facilitates visualization of the flagella. The precipitate from the stain is primarily on the slide rather than the coverslip. Flagella Staining- Principle, Procedure & Result Interpretation Result Interpretation of Flagella Stain 1.Presence or absence of flagella 2.Number of flagella per cell 3.Location of flagella per cell 1.Peritrichous 2.Lophotrichous 3.Polar 4.The amplitude of wavelength 1.Short 2.Long 5.Whether or not “tufted” Limitation Even with a specific stain, visualization of flagella requires an experienced scientist and is India ink (capsule ⚫ The capsule stain employs an acidic stain and a basic stain) stain to detect capsule production. ⚫ Capsules are formed by organisms such as Klebsiella pneumoniae . Most capsules are composed of polysaccharides, but some are composed of polypeptides. PROCEDURE ⚫Place a loopful of India ink on the side of a clean slide ⚫A small portion of the solid culture is suspended in saline on the slide near the ink and then emulsifi ed in the drop of ink, or else, mix a loopful of liquid culture of specimens like CSF with the ink. ⚫Place a clean cover slip over the preparation avoiding air bubbles. ⚫Press down, or blot gently with a fi lter paper strip to get a thin, even film ⚫Examine under dry objectives followed by oil immersion Staining procedure ⚫ Place a loopful of India ink on the side of a clean slide ⚫ A small portion of the solid culture is suspended in saline on the slide near the ink and then emulsifi ed in the drop of ink, or else, mix a loopful of liquid culture of specimens like CSF with the ink. ⚫ Place a clean cover slip over the preparation avoiding air bubbles. ⚫ Press down, or blot gently with a fi lter paper strip to get a thin, even film ⚫ Examine under dry objectives followed by oil immersion Examine under microscope Klebsiella pneomonae Lactophenol Cotton Blue Staining- Principle, Procedure & Result Principle of Lactophenol Cotton Blue (LPCB) Staining Lactophenol Cotton Blue (LPCB) Staining method works on the principle of aiding the identification of the fungal cell walls. •Fungi are eukaryotic organisms with both macroscopic and microscopic characteristics. •The fungal spore cell wall is made up of chitin of which the components of the Lactophenol Cotton Blue solution stains for identification. •The lactophenol cotton blue solution acts as a mounting solution as well as a staining agent. •The solution is clear and blue in color and it is made up of a combination of three main reagents: • Phenol: It acts as a disinfectant by killing any living organisms • Lactic acid: To preserve the fungal structures • Cotton blue: To stain or give color to the chitin on the fungal cell wall and other fungal structures •The stain will give the fungi a blue-colored appearance Lactophenol Cotton Blue Staining- Principle, Procedure & Result Procedure 1.On a clean microscopic glass slide, add a drop of 70% ethanol 2.Add the fungal specimen to the drop of alcohol using a sterile mounter such as an inoculation loop (from solid medium), depending on the sample of use. 3.Tease the fungal sample of the alcohol using a needle mounter, to ensure the sample mixes well with the alcohol. 4.Using a dropper or pipette, add one or two drops of Lactophenol Cotton Blue Solution (prepared above) before the ethanol dries off. 5.Carefully cover the stain with a clean sterile coverslip without making air bubbles to the stain. 6.Examine the stain microscopically at 40X, to observe for fungal spores and other fungal structures. Lactophenol Cotton Blue Staining- Principle, Procedure & Result Results and Interpretation Fungal spores, hyphae, and fruiting structures stain blue while the background stains pale blue. For example, •Aspergillus niger stains the hyphae and fruiting structures a delicate blue with a pale blue background. •Trichophyton mentagrophytes also stains the hyphae and fruiting structures a delicate blue with a pale blue background. Lactophenol Cotton Blue Staining- Principle, Procedure & Result Applications • Used in the identification of suspected fungal samples. • General identification of fungi and its structures. Limitations • It can only be used as a presumptive identification method of fungi which should be followed up with other diagnostic tools such as biochemical and cultural examination. • The components of the solution should be used before expiry, including the use of the solution before it expires. • The solution may disrupt the original morphology of the fungi. • The stain can only be used to identify mature fungi BIOCHEMICAL TESTS ⚫ ⚫ ⚫ ⚫ ⚫ ⚫ ⚫ ⚫ ⚫ ⚫ ⚫ ⚫ ⚫ Indole Methyl Red/Voges Proskauer Citrate Urease Catalase Oxidase Coagulase H2S production in SIM Motility Lactose fermentation Sucrose fermentation Glucose fermentation & gas production Triple Sugar Iron Agar (TSI) test ⚫ Staphylococcus identifi cation tests ⚫ MSA Principle: Indole Test Indole test is performed to determine the ability of the organism to split tryptophan molecule into Indole. Indole is one of the metabolic degradation product of the amino acid tryptophan Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing and deaminating tryptophan with the production of Indole, Pyruvic acid and ammonia. Property it tests for: ⚫ This test is performed to help differentiate species of the family Enterobacteriaceae. Media and Reagents Used: ⚫ Tryptone broth contains tryptophan. ⚫ Kovac’s reagent—contains hydrochloric acid, dimethylaminobenzaldehyde, and amyl alcohol—yellow in color. Indole test • Procedure: ‐Inoculate Tryptone broth with the test organism and incubate for 18 to 24 hrs at 37 c ‐Add 15 drops of Kovac’s reagent down the inner wall of the tube • Interpretation: ‐Development of bright red color at the interface of the reagent and the broth within seconds after adding the reagent is indicative of the presence of Indole and is a positive test Indole Positive: E.coli Proteus vulgaris Indole Negative: Salmonella spp. Klebsiella spp. Enterobacter aerogens M e t h y l Red/Voges‐Proskauer (MR/VP) ⚫ Properties these test for: Both tests are used to differentiate species of the family Enterobacteriaceae. ⚫ Media and Reagents Used: ⚫ Glucose Broth ⚫ Methyl Red indicator for MR test ⚫ Voges Proskauer reagents‐ A: 5% Alpha ‐ Naphthol & ethanol, B: Potassium Hydroxide; (3:1 ratio) & Deionized Water. Principle of MR test: To test the ability of the organism to produce and maintain stable acid end products from glucose fermentation and to overcome the buffering capacity of the system This is a qualitative test for acid production. MR test (contd…) Procedure: ‐ Inoculate the MR/VP broth with a pure culture of the test organism and incubate at 35° for 48 to 72 hrs. Add 5 drops of MR reagent to the broth Result interpretation: ‐ Positive result is red (indicating pH below 6) ‐ Negative result is yellow (indicating no acid production) MR Positive: E. coli MR Negative: Enterobacter aerogenes Enterobacter cloacae Klebsiella spp. Left: negative/Right: positive Voges Proskauer Test (acetoin production) Principle: To determine the ability of the organisms to produce neutral end product acetyl methyl carbinol (acetoin) from glucose fermentation Procedure: Innoculate pure culture of the test organism into MR/VP broth and incubate for 24 hrs at 37°c Aliquot 1 ml of the broth to a sterile test and VP: left + and right – tubeadd 0.6ml of VP(A) followed by 0.2ml of VP(B) Shake the tube gently to expose the medium to Positive atmospheric oxygen and allow the tube to Klebseilla pneumoniae remain undisturbed for 10 to 15 mins Intrepretation: Positive : Pinkish red color at the surface of the medium Negative : Yellow color at the surface of the medium Enterobacter Negative E.coli Citrate Utilization test ⚫ This test is one of several technique used to assist in the identifi cation of enterobacteria. The test is based on the ability of an organism to use citrate as its only sole source of carbon and ammonia as its only source of nitrogen. ⚫ Principle: The test organism is cultured in a medium which contains sodium citrate, an ammonium salt and the indicator bromothymol blue. Growth in the medium is shown by turbidity and a change in colour of the indicator from light green to blue, due to alkaline reaction following citrate utilization. ⚫ Procedure: Inoculum is streaked over the slant of Simmon’s citrate agar in a tube and incubated for 24‐48 hrs. ⚫ Result interpretation: Growth on the slant and change in colour to blue of the medium indicates positive result. Positive: Klebsiella pneumoniae Negative: E. coli Left‐ Positive: Right‐ Negative Oxidation‐ Fermentation (OF) test (Hugh & Leifson) Principle: Oxidation ‐ Fermentation test is used to determine the oxidative or fermentative metabolism of a carbohydrate or its non utilization. Fermentation is a anaerobic process and bacterial fermenters of carbohydrates are usually facultative anaerobes. Oxidation is a aerobic process and bacterial oxidisers are usually strict aerobes Procedures: ‐The method described, sometimes referred to as the Hugh and Leifson test employs a semi‐solid medium in tubes containing the carbohydrate under test (usually glucose) and a pH indicator ‐Two tubes are inoculated and one is immediately sealed with paraffi n oil to produce anaerobic conditions Result interpretation: ⚫ Oxidising organisms, eg Pseudomonas species, produce an acid reaction in the open tube only ⚫ Fermenting organisms, eg Enterobacteriaceae, produce an acid reaction throughout the medium in both tubes ⚫ Organisms that cannot break down the carbohydrate aerobically or anaerobically, eg., Alcaligenes faecalis, produce an alkaline reaction in the open tube and no change in the covered tube Motility Test ⚫ Property it tests for: This test is done to help differentiate species of bacteria that are motile from non ‐ motile. ⚫ Media and Reagents Used: Motility media contains tryptose, sodium chloride, agar, and a color indicator. ⚫ How to Perform Test: Stab motility media with inoculating needle. ⚫ Reading Results: If bacteria is motile, there will be growth going out away from the stab line, and test is positive. If bacteria is not motile, there will only be growth along the stab line. A colored indicator can be used to make the results easier to see. Motility From left to right: + – + Lactose Fermentation ⚫ Property it tests for: This tests for the bacteria’s ability to ferment lactose. ⚫ Media and Reagents Used: Lactose broth contains beef extract, gelatin peptone, and lactose. A phenol red indicator is added to indicate acid production from fermentation. ⚫ How to Perform Test: Inoculate lactose broth with inoculating loop. ⚫ Results ⚫ A positive result is yellow after indicator is added (indicating lactose fermentation) ⚫ A negative result will have no color change or will be reddish. Sucrose Fermentation ⚫ Property it tests for: This test is done to help differentiate species of the family Enterobacteriaceae. This tests the bacteria’s ability to ferment sucrose and production of acid end ‐ product ⚫ Media and Reagents Used: Sucrose broth contains beef extract, gelatin peptone, and sucrose. Phenol red indicator is added to indicate an acid end ‐ product. ⚫ How to Perform Test: Inoculate sucrose broth with inoculating loop. ⚫ Results ⚫ A positive result is yellow after indicator is added (indicating sucrose fermentation) ⚫ A negative result has no color change or is reddish. Glucose Fermentatio n for: & ThisGas ⚫ Property it tests test is done to help differentiate species of the family Enterobacteriaceae. This tests for the bacteria’s abilityProduction to ferment glucose and produce gas and/or an acid end ‐ product.. ⚫ Media and Reagents Used: Glucose broth contains beef extract, gelatin peptone, and glucose. A phenol red indicator is added to indicate an acid end ‐ product. A Durham tube is added to indicate gas production. ⚫ How to Perform Test: Inoculate broth with inoculating loop. ⚫ Results ⚫ A positive result for acid is yellow after indicator is added (indicating glucose fermentation) ⚫ A positive result for gas is a bubble in the Durham tube. ⚫ A completely negative result has no color change or reddish color and no bubble. Sugar Fermentation Test Tube 1: Negative acid /Negative gas Tube 2A: Must incubate longer (ambiguous result) Tube 2B: Positive acid /Negative gas Tube 3A: Positive acid/ Positive gas Triple Sugar Iron Agar (TSI) test ⚫ Principle: TSI agar is used to determine whether a gram negative rod utilizes glucose and lactose or sucrose fermentatively and forms hydrogen sulphide (H2S). TSI contains 10 parts lactose: 10 parts sucrose: 1 part glucose and peptone. Phenol red and ferrous sulphate serves as indicators of acidifi cation and H2S formation, respectively. The formation of CO2 and H2 is indicated by the presence of bubbles or cracks in the agar or by separation of the agar from the sides or bottom of the tube. The production of H2S requires an acidic environment and is indicated by blackening of the butt of the medium in the tube. ⚫ Method: 1. 2. 3. With a straight inoculating wire, touch the top of a well isolated colony. Inoculate TSI by fi rst stabbing through center of the medium to the bottom of the tube and then streaking the surface of the agar slant. Leave the cap on loosely and incubate the tube for 18‐24 hours at 35oC in an incubator. Triple Sugar Iron Agar (TSI) test (contd…) ⚫ Result interpretation: 1. 2. 3. 4.  Alkaline slant/no change in the butt (K/NC) = Glucose, lactose and sucrose non ‐ utilizer (alkaline slant/alkaline butt) [figure: 1(d)] Alkaline slant/acid butt (K/A) = Glucose fermentation only. [figure: 1(b)] Acid slant/acid butt (A/A), with gas production = Glucose, sucrose, and/or lactose fermenter. [figure: 1(a)] Alkaline slant/acid butt (K/A), H2S production = Glucose fermentation only. [figure: 1(c)] Quality control: A/A, with gas: E. coli K/A, H2S: Salmonella typhi K/NC: Pseudomonas aeruginosa a b Figure (1): TSI results c d Mannitol Salt Agar (MSA) ⚫ Property it tests for: This tests for the bacteria’s ability to tolerate 7% salt concentration and ferment mannitol. The media is selective because it selects for salt tolerant bacteria. ⚫ Media and Reagents: MSA media contains nutrient agar, mannitol, 7% sodium chloride and phenol red indicator. ⚫ How to Perform Test: Inoculate an MSA plate using streak plate method and incubate 24‐48 hours. MSA Results ⚫ Reading Results: ⚫ If the organism is tolerant to salt it will grow. ⚫ If the organism is not tolerant to salt it will not grow. ⚫ If the salt tolerant organism can ferment mannitol then there will be yellow zones around the colonies. ⚫ If the salt tolerant organism cannot ferment mannitol then the media will remain pink. Growth with no mannitol fermentation. Growth with + mannitol fermentation. Enzyme-based biochemical tests ⚫ Catalase test ⚫ Oxidase test ⚫ Urease test ⚫ Coagulase test ⚫ Nitrate reduction Catalase test Principle: This test demonstrates the presence of enzyme catalase in the organism. The enzyme catalase mediates the breakdown of hydrogen peroxide (H2O2) into oxygen and water. The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide (30% for slide test), and the rapid effervescence of O2 bubbles occurs. The lack of catalase is indicated by a lack of bubble production.lase 2H2o2 catalase 2H2o + o2 (gas bubbles) Catalase test…. • Catalase is present in most cytochrome containing aerobic and facultative anaerobic bacteria (except streptococcus spp). • Hydrogen peroxide forms as one of the oxidative end product of aerobic carbohydrate metabolism. If this is allowed to acculmulate in the bacterial cells it becomes lethal to the bacteria • Catalase thus helps in converting H2 o2 to H 2 o and o2 • Optimal pH for catalase action is 7. Catalase test…. Reagents: • 3% hydrogen peroxide stored in dark brown bottle under refrigeration • 18 to 24 hrs culture of the organism to be tested Control organisms used: Positive control ‐ Staphylococcus aureus Negative control – Streptococcus spp. Catalase test…. Positive • Micrococcus • Staphylococcus • Bacillus • Listeria monocytogenes • Enterobacteriacae • Gonococcus & Meningococcus • Vibriocholerae • Negative • Streptococcus • Clostridium Oxidase Test Principle Oxidase test is used to determine the presence of bacterial cytochrome oxidase enzyme using the oxidization of the substrate “tetramethyl ‐p ‐ phenylenediamine dihydrochloride” to indophenol a dark purple colored end product. A positive test (presence of oxidase) indicated by the development of a dark purple colour. No colour development indicates a negative test and the absence of the enzyme. Oxidase Test….   Cytochromes are iron containg hemoprotiens and in aerobic respiration they transfer electrons(H) to oxygen to form water. The reagent acts as an artificial electron acceptor substituting the oxygen. In the reduced stage dye is colorless , but in the presence of enzyme cytochrome oxidase dye is oxidised to indophenol blue Methods 1. Moist fi lter paper method Quality controls Positive control‐ Pseudomonas spp Negative control – E. coli 2. Direct plate method Oxidase Test….. Positive Negative • Pseudomonas spp. • Aeromonas spp. ⚫ Enterobacteriaceae • Vibrio spp. • Alcaligenes spp. • Neisseria spp. • Haemophilus sps ⚫ Acenitobacter spp. Urea Hydrolysis (Urease test) ⚫ Property it tests for: This test is done to determine a bacteria’s ability to hydrolyze urea to make ammonia using the enzyme urease. ⚫ Media and Reagents Used: Stuarts Urea broth (pH 6.8) contains a yeast extract, monopotassium phosphate, disodium phosphate, urea, and phenol red indicator. ⚫ Principle To determine the ability of the organism to split urea forming 2 molecules of ammonia by the action of the enzyme Urease with resulting alkalinity ⚫ How to Perform Test: Inoculate Urea broth with inoculating loop. Reading Results: ⚫ Urea broth is a yellow‐orange color. The enzyme urease will be used to hydrolyze urea to make ammonia. If ammonia is made, the broth turns a bright pink color, and is positive. If test is negative, broth has no color change and no ammonia is made. ⚫ Figure in the right shows negative (left) and Positive (right) results. Coagulas ⚫ Principle: e test This test is used to differentiate Staphylococcus aureus (positive) from ‐ coagulase negative Staphylococci. S. aureus produces two forms of coagulase: bound and free. ‐ Bound coagulase or clumping factor, is bound to the bacterial cell wall and reacts directly with fi brinogen. When a bacterial suspension is mixed with plasma, this enzyme causes alteration in fi brinogen of the plasma to precipitate on the staphylococcal cells, causing the cells to clump. ‐ Free coagulase is produced extra‐cellularly by the bacteria that causes the formation of a clot when S. aureus colonies are incubated with plasma. ⚫ Method: A. ‐ ‐ ‐ ‐ B. ‐ ‐ ‐ Slide test: (for bound coagulase) Place a drop of coagulase plasma on a clean, dry glass slide. Place a drop of distilled water or saline next to the drop of plasma as a control. With a loop or wooden stick, emulsify a portion of the isolated colony being tested in each drop. Mix well and rock the slide gently for 5 to 10 seconds. Tube test: (for free coagulase) Emulsify several colonies in 0.5 ml of rabbit plasma (with EDTA) to give a milky suspension. Incubate tubes at 35oC in ambient air for 4 hrs. Check for clot formation. If negative at 4 hrs, incubate at room temperature overnight and check again for clot formation. Coagulase Results ⚫ Reading Results: A. Slide test: ‐ Positive: Macroscopic clumping in 10 seconds or less in coagulated plasma drop and no clumping in saline or water drop. ‐ Negative: No clumping in either drop. ‐ Note: All negative slide tests must be confi rmed using the tube test. B. Tube test: ‐ Positive: Clot of any size (a) ‐ Negative: No clot (b) a Coagulase Positive : Staphylococcus aureus Coagulase negative: Staphylococcus b Nitrate reduction test Principle: This test is used to determine the ability of the organism to reduce nitrate to nitrites or fee nitrogen gas. The reduction of nitrate to nitrite is detected by adding sulphanilic acid and alpha ‐ naphthylamine. The sulphanilic acid and nitrite reacts to form a diazonium salt which then reacts with alpha ‐ naphthylamine to produce a red, water soluble azo‐dye. Purpose: ⚫ Aid in the species deferentiation of i) Haemophilus duceryi(‐) and Haemophilus vaginalis(‐) from other Haemophillus spp. ii) Neisseria mucosa(+) from other Neisseria spp ⚫ Aid in the identifi cation of Enterobacteriaceae(+) Nitrate reduction test…. Procedure: • In order to determine if a bacteria can reduce nitrate, the test organism is inoculated into nitrate broth [an undefi ned medium that contains large amounts of nitrate (KNO3)]. After incubation, alpha ‐ naphthylamine and sulfanilic acid are added . These two compounds react with nitrite and turn red in color, indicating a positive nitrate reduction test • If there is no color change at this step, nitrite is absent. If the nitrate is unreduced and still in its original form, this would be a negative nitrate reduction result. However, it is possible that the nitrate was reduced to nitrite but has been further reduced to ammonia or nitrogen gas (which evolved out). This would be recorded as a positive nitrate reduction result Nitrate reduction…. • To distinguish between these two reactions, zinc dust must be added. Zinc reduces nitrate to nitrite. If the test organism did not reduce the nitrate to nitrite, the zinc will change the nitrate to nitrite. The tube will turn red because alpha ‐ naphthylamine and sulfanilic acid are already present in the tube • Thus a red color after the zinc is added indicates the negative nitrate reduction test. Nitrate reduction test…. Negative Negative Nitrate not reduced Posiitive Nitrate reduced to nitrite Positive Nitrate reduced to NH3 or N2 gas, nitrite absent Nitrate not reduced Nitrate reduction test…. Addition of Zn dust or • Principle : Bile-esculin test is based on the ability of the group D streptococci and Enterococcus species, to hydrolyze esculin in the presence of bile. • Bile esculin agar contains bile salts to inhibit the growth of gram positive organisms other than enterococci and group D streptococci. • It also contains nutrients, esculin, and ferric citrate. • When an organism hydrolyzes the glycoside esculin to form esculetin and dextrose, the esculetin reacts with the ferric citrate to produce a dark brown or black phenolic iron complex. Bile Esculin Test Gram positive fl owchart present Gram negative fl owchart CULTURE MEDIA Culture media and their preparation  Medium (media pl.) is the substance which provides nutrients for the growth of microorganisms.  The nutrients on which microorganisms are cultivated called culture medium (pl. culture media).  Culture media vary in their form and composition determined by the species to be cultivated. There is no single medium which can support the growth of majority of microbes  It is used to grow, transport and store microorganisms. History • Louis Pasteur used simple broths made up of urine or meat extracts. • Robert Koch realized the importance of solid media and used potato pieces to grow bacteria. It was on the suggestion of Fannie Eilshemius, wife of Walther Hesse (who was an assistant to Robert Koch) that agar was used to solidify culture media. • Before the use of agar, attempts were made to use gelatin as solidifying agent. • Gelatin had some inherent problems; it existed as liquid at normal incubating temperatures (3537oC) and was digested by certain bacteria. • History • Louis Pasteur used simple broths made up of urine or meat extracts. • Robert Koch realized the importance of solid media and used potato pieces to grow bacteria. It was on the suggestion of Fannie Eilshemius, wife of Walther Hesse (who was an assistant to Robert Koch) that agar was used to solidify culture media. • Before the use of agar, attempts were made to use gelatin as solidifying agent. • Gelatin had some inherent problems; it existed as liquid at normal incubating temperatures (3537oC) and was digested by certain bacteria. • Composition of culture media • Bacteria infecting humans (commensals or pathogens) are chemoorganoheterotrophs. • When culturing bacteria, it is very important to provide similar environmental and nutritional conditions that exist in its natural habitat. • Hence, an artificial culture medium must provide all the nutritional components that a bacterium gets in its natural habitat. • Most often, a culture medium contains water, a source of carbon & energy, source of nitrogen, trace elements and some growth factors. • Besides these, the pH of the medium must be set accordingly. • Some of the ingredients of culture media include water, agar, peptone, casein hydrolysate, meat extract, yeast extract and malt extract. Classification Bacterial culture media can be classified in at least three ways; • Based on consistency (liquid, semisolid or solid and biphasic) • based on nutritional component (simple, complex and synthetic (or defined)), and • based on its functional use (basal media, enriched media, selective/enrichment media, indicator/differential media, transport Liquid media Liquid media are sometimes referred as “broths” (e.g nutrient broth). In liquid medium, bacteria grow uniformly producing general turbidity. Growth is described characterized by turbidity Liquid media tend to be used when a large number of bacteria have to be grown. These are suitable to grow bacteria when the numbers in the inoculum is suspected to be low. Inoculating in the liquid medium also helps to dilute any inhibitors of bacterial growth. This is the practical approach in Sterilization and Disinfectio n Definitions Sterilizatio removal or killing of all living n microorganisms including bacteria and their spores Disinfectio removal or killing of diseasen causing microorganisms (not necessarily all microorganisms) Methods of sterilization: There are two methods of sterilization: APhysical methods: 1. Sterilization by Heat 2. Sterilization by Filtration 3. Sterilization by Irradiation BChemical methods Hea t Sterilizati on Physic al Filtratio n Chemic al Irradiati on Sterilization by heat • Heat is the most practical, reliable, and inexpensive method of sterilization. • It is used for sterilization of objects and materials that can withstand high temperatures. • It can be either: • Dry heat • Moist Heat Methods of Sterilization Red Heat Dry Heat Heat Sterlizati on Moist Heat Flamin Incinerati g on Hot Air Oven Below 100 °C  Pasteurization At 100 °C  Boiling Above 100 °C  Autoclaving 1. Red Heat Principal: Used for: Holding object in Bunsen flame till they become red hot. Sterilization of: • Bacteriological loops • Tips of forceps 2. Flaming Principal: Passing the object through the flame of Bunsen burner without heating to redness. Used for: • glass slides Sterilization • mouth of of: culture tubes. 3. Incineration Principal: Used for: Infective materials is converted to sterile ash by burning in incinerator Destruction of contaminated disposable materials (waste) 4. Hot air oven Principal • Articles to be sterilized are exposed to high temperature in an electrically heated oven. • Even distribution of heat throughout the chamber is achieved by a fan. Holding time: • 160°C for two hour • 180°C for one hour 4. Hot air oven Used for Sterilization of: • All glasses: test tubes, Petri dishes, flasks, pipettes. • Instruments: as forceps, scalpels, scissors • Dry material in sealed containers as fat, oils, powder. Moist Heat Can be used at • below 100 temperatures: °C • At 100 °C • Above 100 °C different  Pasteurizatio n  Boiling  Autoclave 1. Pasteurization (Below 100) Principal: Pasteurization is a process of heating a liquid to a specific temperature for a definite length of time and then cooling it immediately. Used for: Pasteurization is commonly used in milk processing. 2. Boiling (At 100 °C) Principal: Boiling in water for fifteen minutes will kill most vegetative bacteria and inactivate viruses. However boiling is ineffective against many bacterial and fungal spores. 3. Autoclaving Principal: • When the pressure is increased inside a closed container, the temperature at which water boils exceeds 100°C. • At double atmospheric pressure the temperature of the steam reaches 121°C. • Autoclaving is the most reliable method of sterilization that kills all kinds of bacteria and spores. 3. Autoclaving Temperature of sterilization: - 121C for 20 – 30 minutes. Used for sterilization of: • Culture media. • Surgical supply e.g. dressing, and surgical instruments. Filtration • Filtrations is used to sterilize liquids that would be damaged by heat as sera, antibiotic solutions and vaccines. Sterilization by irradiation Irradiatio n Ionizing Gam ma rays NonIonizi ng Ultraviol et (UV) rays Ultraviolet radiation Used for. 1. Sterilization of operating theatre 2. Sterilization of the interiors of biological safety cabinets Ionizing irradiation (gamma rays) • Used for sterilization of an article not stand heat as rubber catheters, gloves, plastic syringes. Chemical methods of Sterilization • Disinfectant: • Are chemical materials used for sterilization but are toxic to the human tissues and cells. • Antiseptics: • Are chemicals for sterilization but not toxic to the human body e.g. "mouth gargles". Examples of disinfectant and antiseptics There are a number of chemicals that can act as disinfectants or antiseptics. These include: • Phenol and its derivatives e.g. Dettol. • Halogens e.g. Chlorine, Tincture iodine. • Alcohols e.g. ethyl alcohol. • Aldehydes e.g. glutaraldehyde (Cidex), Formalin. • Quaternary Ammonium Compounds (Cationic detergents). Inoculation techniques A-Pour Plate: In this method, fixed amount of inoculum from a broth/sample is placed in the center of sterile Petri dish using a sterile pipette. Molten cooled agar (approx. 15mL) is then poured into the Petri dish containing the inoculum and mixed well. After the solidification of the agar, the plate is inverted and incubated at 37°C for 24-48 hours. B. Spread Plate: Introduce an inoculum onto the surface of Petri dish C. Streak Plate In this method, inoculating loop is used to transfer bacterial colonies/cells onto a solid medium and further used to spread cells onto a thin line D. Stabbing technique In this method, an innoculating loop with desired organism is stabbed unto the growth medium either at the surface or deep into the medium. Organism grows at the point of stab. -Microbiologists typically count plates with 30–300 colonies. Samples with too few colonies (<30) do not give statistically reliable numbers, and overcrowded plates (>300 colonies) make it difficult to accurately count individual colonies. Measuring Microbial Growth Direct Methods of Measurement 1. Plate count Physically count the number of colonies growing on the culture medium 2.Filtration -Used to measure small quantities of bacteria. • Example: Fecal bacteria in a lake or in ocean water. A large sample (100 ml or more) is filtered to retain bacteria. Filter is transferred onto a Petri dish. Incubate and count colonies. 3- Direct Microscopic Count Involves transferring a known volume of a culture to a calibrated slide and counting the cells under a light microscope. The calibrated slide is called a Petroff- Hausser chamber Measuring Microbial Growth Indirect Methods of Measurement 1 Turbidity: As bacteria multiply in media, it becomes turbid. Spectrophotometer is used to measure turbidity 2.Metabolic Activity -As bacteria multiply in media, they produce certain products such as Carbon dioxide and Acids. -Measure metabolic products. 3.Dry Weight (Biomass concentration :It is the dry weight of cells Per unit volume of culture). -Bacteria in liquid media are centrifuged. - Resulting cell pellet is weighed. Serial Dilution The serial dilution of a culture is an important first step before proceeding to either the pour plate or spread plate method. The goal of the serial dilution process is to obtain plates with CFUs in the range of 30–300, and the process usually involves several dilutions in multiples of 10 to simplify calculation. Serial dilution involves diluting a fixed volume of cells mixed with dilution solution using the previous dilution as an inoculum. The result is dilution of the original culture by an exponentially growing factor.