Identification of bacteria

bacterial staining and identification

1. Identification of Bacteria By: Mrs. Mali Dhanashri R. Assistant professor, GES’s Sir Dr. M. S. Gosavi College of Pharmaceutical Education and Reseach

2. Identification is the process of determining to which established taxon a new isolate or unknown strain belongs. Identification of unknown bacterial culture is one of the major responsibilities of a microbiologist Samples of blood, tissue, water, food and cosmetics are examined daily in laboratories throughout the world for the presence of contaminants and pathogenic microorganisms. Pharma industries and research institutes are constantly screening soil, water, marine samaples to isolate new antibiotics, enzymes and vitamins producing microorganisms. Bergey’s Manual of Systemic Bacteriology (BMSB) has been the official, internationally accepted reference for bacterial classification. In the current edition of Bergey’s manual, bacteria is classified into 33 groups called sections and contain information of 4000 bacterial species.

3. Importance of identification Medical diagnostics & clinical significance — identifying a pathogen isolated from a patient. Food & brewage industries — identifying a microbial contaminant responsible for food spoilage and fermentation. Research setting — identifying a new isolate which carries out an important process.

4. IDENTIFICATION METHODS The methods fall into three categories: Phenotypic/Morphology (micro and macroscopic) Immunological/Serological analysis Genetic techniques/Molecular method

5. Staining Techniques To study size, shape, arrangement and properties and different specific groups of microorganisms, Biological stains are used. Stains is an organic compounds containing a benzine rings with chromophore and auxochrome group. Different types of staining techniques are used to study the morphological and structural properties of microorganisms.

6. Types of staining

7. Dyes Basic dyes : Methylene blue, Basic fuchsin , Crystal violet, Safranine , Malachite green. Have positively charged groups (usually from penta-valent nitrogen). Generally sold as chloride salts. Basic dyes bind to negatively charged molecules such as nucleic acids, many proteins and surfaces of bacterial and archeal cells. Used for Positive staining

8. Acidic dyes : Eosin , Rose Bengal , Nigrosin ( indian ink ), Congo red and Acid fuchsin possess groups such as carboxyls (-COOH) and phenolic hydroxyls (-OH). Acidic dyes, in their ionized form, have a negative charge and bind to positively charged cell structures. Used for negative staining Neutarl dyes: Both positively and negatively charged ions impart diiferent colour to different component E.g. Leishman’s stain, Wrights stain.

9. Smear Preparation Microbial smear : It is a very small amount of microbial growth spread on a clean slide and drying by air . The purpose of making a smear is to fix the bacteria onto the slide and to prevent the sample from being lost during a staining procedure. A smear can be prepared from a solid or broth medium. The preparation of a smear is required for many laboratory procedures, including the Gram-stain.

10. Procedure Place one needle of solid bacterial growth or two loops of liquid bacterial growth in the center of a clean slide. If working from a solid medium, add one drop (and only one drop) of water to your specimen with a water bottle. If using a broth medium, do not add the water. Now, with your inoculating loop, mix the specimen with the water completely and spread the mixture out to cover about half of the total slide area. Place the slide on a slide warmer and wait for it to dry. The smear is now ready for the staining procedure.

11. Smear selection: If selected smear is embedded in paraffin, it is necessary to remove the paraffin so that a watery stain may penetrate the bacterial cell. The paraffin is removed with xylene, the xylene is then removed with alcohol and the alcohol is replaced with water. The staining is then done.

12. Smear fixation: Fixation of bacterial cell on slide. Heat Fixation: The process of passing the smear after drying several times over benzene burner to fix the microbes on slide and prepare it for staining . Allow the slide to cool before staining Methanol Fixation: Flood the smear with methanol for one minute. Drain the slide and allow to dry.

13. SIMPLE STAINING/Monochrome Stains Used only single stain Ex: Any one at a time- Methylene blue(2-3mins), Crystal violet(1- 2mins), Carbol fuchsin (15-30secs), Safranin(1mins), Malachit green(1-2mins), etc. Used to study Size, shape and bacterial cell arrangements. Basic stains with a positively charged chromogens (any substance that can become a pigment or coloring matter, as a substance in organic fluids that forms coloured compounds when oxidized, or a compound, not itself a dye, that can become a dye) are used. Purpose: To elucidate the morphology and arrangement of bacterial cells.

14. PROCEDURE Select oil/grease free slide. Do it by washing with detergent and wiping the excess water and then dry the slide by passed through flame. These slide is allowed to air dry and smear of sample is applied. After air drying these slide is rapidly passed through a flame for three to four times for heat fixation. After heat fixation the slide is flooded with a particular stain and these stain is allowed to react for two-three minutes. Further the slide is washed under running distilled water. The slide is air dried and watched under oil immersion microscope.

15. Mechanism behind the simple staining A stain has a ability to bind a cellular component .These abilities depend upon the charges present on cellular component and charges present on chromophore group of stain. Bacteria has large number of carboxyl group on its surface and these carboxyl group has negative charge. When these carboxyl group carry out ionization reaction it shows COO – and H + COOH Ionisation COO – + H + In nature, these H+ ions (unstable) are present on cell surface and further replaced by other positively charged ions like Na + or k + and H + bonds with oxygen to form water.

16. NEGATIVE STAINING Colouring the Background of Object Not stain but made visible against dark background Bacteria are mixed with acidic stains such as Eosin or Nigrosin that provide a uniformly coloured background against which the unstained bacteria stand out in contrast. Useful to observed bacteria that are difficult to stain ( Spirili & spirochetes– Trepanoma palladium, Borrelia burgdorferi, Leptospira) and in demonstration of bacterial capsule. Acidic stains has negative charge; therefore, it doesn’t combine with negatively charged of bacteria cell surface.

17. Positive staining Negative staining Simple positive staining: all bacteria are colored, Simple negative staining: background is dark, bacteria are without any color

18. Advantages over Simple: Natural size and shape of microorganism can be seen Heat fixation is not required Doesn't need physical and chemical treatment. It is possible to observe bacteria that are difficult to stain.

19. Differential staining techniques The differential staining techniques are based on the application of a set of several different dyes which react differently with different types of microorganism. Hence, they can be used to distinguish among them. An example is the Gram staining technique. This procedure separates bacteria into two groups: Gram positive bacteria and Gram negative bacteria.

20. GRAM STANING It is not only reveals the size and shape of bacteria but also used to differentiate bacteria into Gram positive and Gram negative cells. Hence, called differential staining. It is first and usually the only method employed for the diagnostic identification of bacteria in clinical specimens. Provides more information about the characteristics of the cell wall (Thickness). A stain is a chemical substance that adheres to a cell, giving the cell colour .

21. Principle Dr. HANS CHRISTIAN GRAM: Danish bacteriologist noted for his development of the Gram stain in 1884. Violet dye and the iodine combine to form an insoluble, dark purple compound in the bacterial protoplasm and cell wall. This compound is dissociable in the decolourizer , which dissolves and removes its two components from the cell. But the removal is much slower from Gram-positive than from the Gram-negative bacteria, so that by correct timing the former stay dark purple whilst the latter become colourless . The difference between the two types of bacteria is that the Gram positive have thicker and denser peptidoglycan layers in their cell walls, which makes them less permeable to the stain than those of the Gram negative bacteria. The iodine has a critical role in enhancing this difference. It seems to bind temporarily to the peptidoglycan and make it even less permeable to the dye.

22. Procedure: Thin smear of bacterial culture was made on clean glass slide Air dried and heat fixed Smear was covered with crystal violet for 30 seconds Slide was washed with distilled water Smear was covered with Grams iodine solution for 60 seconds Slide was washed with 95% ethyl alcohol and then distilled water Again the smear was covered with safranin for 30 seconds Washed with distilled water and blot dried Air dried and observed under microscope

23. Gram-positive bacteria appear blue or purple E.g. Staphylococcus aureus, Streptococcus pyogenes, Corynebacterium diphteriae Gram-negative cells will appear pink to red E.g.Neisseria meningitidis, Escherichia coli, Pseudomonas aeruginosa

24. ACID FAST STAINING Another widely used differential staining procedure in bacteriology developed by Ehrlich in 1882. Also known as Ziehl – Neelsen staining. Some bacteria(specially M. Tuberculosis, Nocardia asteroides , Actinomycetes, and M. leprae ) resist toward the gram staining or other staining due to high lipid content in the cell wall, hence they are called ACID FAST BACTERIA. 5% aquouse phenol and heat(physical intensifier) are used to enhance the penetration of primary stain(chemical intensifier)

25. Some bacteria produce the waxy substance  mycolic acid  when they construct their cell walls. Mycolic acid acts as a barrier, protecting the cells from dehydrating, as well as from phagocytosis by immune system cells in a host. This waxy barrier also prevents stains from penetrating the cell, which is why the Gram stain does not work with mycobacteria such as  Mycobacterium , which are pathogens of humans and animals .

26. Procedure : Place a drop of NaCl onto the glass slide. Using a sterilized and cooled inoculation loop, obtain a very small sample of a bacterial colony. Gently mix the bacteria into the NaCl drop. Let the bacterial sample air dry Using slide holder, pass the dried slide through the flame of Bunsen burner 3 or 4 times, smear side facing up. Flood slides with Kinyoun carbolfuchsin (or Ziehl-Neelsen Carbol Fuchsin (ZNCF) for 5 minutes. Rinse gently with water until the water flows off clear. Flood slides with acid-alcohol (3% HCl in ethanol) for 3~5 seconds. Rinse gently with water until the water flows off clear. Flood slides with methylene blue for 1 minutes. Rinse gently with water until the water flows off clear. Allow slides to air dry before viewing

27. acid fast bacteria appear bright red E. g. Mycobacterium tuberculosis, Mycobacterium avium non-acid fast bacteria appear blue E.g. Escherichia coli, Staphylococcus aureus, etc .

28. Endospore Stain Endospores are dormant forms of living bacteria Mature endospores are highly resistant to environmental conditions such as heat and chemicals. Because the endospore coat is highly resistant to staining, a special method was developed to make them easier to see with a brightfield microscope. This method, called the  endospore   stain , uses either heat or long exposure time to entice the endospores to take up the primary stain, usually a water soluble dye such as malachite green since endospores are permeable to water. Following a decolorization step which removes the dye from the vegetative cells in the smear, the counterstain safranin is applied to provide color and contrast.

29. SCHAEFFER AND FULTON METHOD: Spore staining, Malachit green (primary stain) and counter stain(safranin). Thin smear of bacterial culture was made on clean glass slide Air dried and heat fixed Smear was covered with malachite green Slide was steamed for 5 minutes by adding more stain to the smear for time to time Slide was washed slowly under tap water Again the smear was covered with safranin for 30 seconds Smear was washed with distilled water and air dried Observed under microscope

30. Endospores can also be directly observed in cells by using phase contrast microscopy

31. Capsule staining Bacterial capsules play a role in adherence, protection (they inhibit ingestion and killing by phagocytes), securing nutrients, and cell-to-cell recognition. The capsule is non-ionic, so that the dyes commonly used will not bind to it. Two dyes, one acidic and one basic, are used to stain the background and the cell wall, respectively. Negative staining methods contrast a translucent, darker colored, background with stained cells but an unstained capsule. The background is formed with india ink or nigrosin or congo red. A positive capsule stain requires a mordant that precipitates the capsule. By counterstaining with dyes like crystal violet, methylene blue or carbolfuchsin , bacterial cell wall takes up the dye. Capsules appear colourless with stained cells against dark background.

32. Procedure Using sterile technique, add a loopful of bacterial culture to tube with 1 ml NaCl. Add one drop of carbolfuchsin into the tube and mix gently. Heat the mixture under flame 1min. Place a drop of mixture onto the glass slide. Place a drop of nigrosin onto the same glass slide next to drop of mixture of bacteria and the dye. Use the other slide to drag the nigrosin -cell mixture into a thin film along the first slide. Allow to air dry for 5-7 minutes (do not heat fix). Examine the smear microscopically (100X) for the presence of encapsulated cells as indicated by clear zones surrounding the cells.

33. Encapsulated bacteria: Clear halos (capsules) are observed around pink bacteria against dark background E.g. Bacillus anthracis, Klebsiella pneumoniae, Streptococcus pneumoniae, Neisseria meningitidis, Clostridium perfringens; Bacteria without capsules: pink bacteria with no clear halos E.g. Neisseria gonorrhoreae , etc.

34. Biochemical Tests The microbe is cultured in a media with a special substrate and tested for an end product. Prominent biochemical tests include:  Enzymes  Carbohydrate fermentation  Acid production  Gas production  Sensitivity to drugs

35. Biochemicals…. Enzymes Catalase test Oxidase test Urease test Coagulase test

36. CATALASE TEST Catalase is present in most 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 accumulate in the bacterial cells it becomes lethal to the bacteria Catalase thus helps in converting H 2 O 2 to H 2 O and O 2 The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide and the rapid effervescence of O 2 bubbles occurs.

37. Procedure Nutrient agar medium was prepared The medium was poured into culture tubes and flasks It was sterilized by autoclaving at 151b pressure for 15 minutes The nutrient agar slants were inoculated with test organisms An inoculated nutrient agar slant was kept as control The cultures were incubated at 35°C and 3-4 drops of hydrogen peroxide was added on the growth of each slant culture The culture was observed for the appearance or absence of gas bubbles.

38. Positive  Micrococcus  Staphylococcus  Bacillus  Listeria monocytogenes  Enterobacteriacae  Gonococcus & Meningococcus  Vibriocholerae  Pseudo/Aero/ Plesiomonas

39. OXIDASE TEST This test depends on the presence of cytochrome oxidase in bacteria  Procedure- Place a piece of filter paper in petri dish and add 3 drops of freshly prepared oxidase reagent (1% solution of tetramethyl-p-phenylene diamine)  Using a sterile glass rod or toothpick, remove a colony of test organisms from a culture plate and smear it on the filter paper  Oxidase positive organisms give blue/ dark purple color within 5-10 seconds, and in oxidase negative organisms, color does not change.

40. Positive  Pseudomonas spp.  Aeromonas spp.  Vibrio spp.  Alcaligenes spp.  Neisseria spp.  Haemophilus sps

41. Coagulase test  This test is used to differentiate Staphylococcus aureus (positive) from coagulase negative Staphylococci  When a bacterial suspension is mixed with plasma, this enzyme causes alteration in fibrinogen.  leading to precipitation on the staphylococcal cells, causing the cells to clump.  Slide test : Positive when there is Macroscopic clumping in 10 seconds or less in a plasma drop and no clumping in a saline or water drop.  Tube test: Positive when there is a Clot of any size

42. Urease test  Some bacteria produce urease an enzyme that hydrolyzes urea into ammonia and carbon dioxide.  The test for urease production relies on the fact that the ammonia produced upon hydroysis is alkaline.  The test organism is inoculated into a urea broth that contains phenol red, a pH indicator, and has a pH of 6.8. At this pH phenol red is salmon color. However, when the pH rises above 8.1 phenol red turns a cerise (hot pink) color.  The urease test is useful for differentiating Salmonella which is urease negative , from Proteus which is urease positive .

43. Urease agar medium was prepared and sterilized using autoclave The medium was allowed to solidify in the slanting position to form a slope The slants were inoculated with test organism The tubes were incubated at 37°C for 24 to 48hrs The slants were observed for colour

44. IMViC test: These are a group of individual tests used in microbiology lab testing to identify an organism in the  coliform group. Each of the letters in “ IMViC ” stands for one individual test. Indole : Break down the amino acid Tryptophan Methyl Red: Glucose oxidation Voges-Proskauer : Production of neutral end products Citrate : Citrate fermentation

45. The lower case " i " is merely for "in" as the Citrate test requires coliform samples to be placed "in Citrate".  Triple Sugar Test (TSI), Urease Test and   Malonate Test are also included in this test. Some microorganism are differentiate on the basis of enzyme- catalysed metabolic reactions Presence and absence of certain enzymes, intermediately metabolites end products often give valuable information in identifying and classifying microorganism. These tests are useful in distinguishing members of Enterobacteriaceae (Aerobic, non-acid fast, gram negative bacilli found in human and other animal intestine.

46. Indol Production test Some bacteria posses tryptophanase enzyme. The enzyme is capable of converting tryptophan (an amino acid) to indole and pyruvic acid, ammonia. Pyruvic acid can be converted to energy or used to synthesize other compounds required by the cell. Indole reacts with added Kovac’s reagent to form rosindole dye which is red in color ( indole +). Principle Reaction:

47. Indol production is detected by inoculating the test microorganisms into peptone water and incubating it at 37°C for 2-5days. Development of cherry red colour at the interface of the reagent and the broth, within seconds after adding the Kovacs’ reagent indicates the presence of indole and the test is positive. If no colour change is observed, then the test is negative and so organisms are not capable of producing indol .

48. Procedure: 1% tryptone broth was prepared and sterilized using autoclave at 151bs for 15 minutes The tryptone broth was inoculated with test organism and an uninoculated tube was kept as control The tubes were incubated at 35°C for 48hours; 1ml of Kovac s reagent was added 48hrs of incubation The tubes were shaken gently after intervals of 10 to 15minutes The tubes were allowed to stand for few minutes to permit the reagent to come to the top. The tubes were observed for colour produced in the top layer

49. Methyl red (MR) Test Used to detect the production of acid during fermentation of glucose. Principle : This is to detect the ability of an organism to produce and maintain stable acid end products from glucose fermentation . Some bacteria produce large amounts of acids from glucose fermentation that they overcome the buffering action of the system. By production of acid, pH of the medium falls and maintained below 4.5 Methyl red is a pH indicator (red at pH less than 4.4 and yellow at a pH greater than 6 ). Methyl red test and Voges-Proskauer test  both are done in methyl red– Voges-Proskauer (MR-VP) broth, but the reagents that are added varies according to the  test.

50. Mainly used to differentiate between E.coli and E. aerogen Principle Reactions:

51. Procedure: MRVP broth ( Glucose phosphate peptone water) was prepared and sterilized using autoclave 5ml of the broth was poured into each tube The tubes were inoculated with test organism All the tubes were incubated at 25°C for 48hrs 5 drops of methyl red indicator was added to the tubes of each set The change in color of methyl red was observed for MR test A red colour indicates that glucose has been oxidized. Methyl red positive: Red colour (Acid production )- E.coli Methyl red negative: No colour (No acid production )- E . aerogen

52. Voges-Proskauer Test Used to determine the ability of microbes to produce non-acidic or neutral end products (acetyl methyl corbinol - acetoin , 2,3-butanediol and Diacetyl ). Principle: Acetyl-methyl carbinol ( acetoin ) is an intermediate in the production of butylene glycol. In this test two reagents, 40 % KOH and alpha-naphthol are added to test broth after incubation and exposed to atmospheric oxygen. If acetoin is present, it is oxidized in the presence of air and KOH to diacetyl . Diacetyl then reacts with guanidine components of peptone, in the presence of alphanaphthol to produce red color. Role of alpha-naphthol is that of a catalyst and a color intensifier.

53. MR-VP broth is a combined medium used for two tests— Methyl Red and Voges-Proskauer . This test is characterizes E.aerogen In the alkali medium and oxygen, the amount of acetyl methyl carbinol present in the medium is oxidised to diacetyl which react with the peptone of the broth to give a red colour . Principle Reactions:

54. Procedure: MRVP broth (Glucose phosphate peptone water) was prepared and sterilized using autoclave 5ml of the broth was poured into each tube The tubes were inoculated with test organism All the tubes were incubated at 25°C for 48hrs 12 drops of VP reagent -1 and 2-3 drops of VP reagent- II was added to the other set of tubes The tubes were gently shaken for 30 seconds with the caps off to expose the media to oxygen The tubes were kept aside for 15-30minutes and observed for change in colour for the VP test Klebsiella pneumoniae: Positive Escherichia coli: Negative

55. Citrate Utilization Test Used to determine if an organism is capable of fermenting citrate as the sole source of carbon for growth. It indicated by the production of turbidity in the medium. The ability of an organism to utilize citrate occurs via the enzyme citrase .

56. Principle: Bacteria are inoculated on a medium containing sodium citrate and a pH indicator bromothymol blue. Utilization of citrate involves the enzyme citritase , which breaks down citrate to oxaloacetate and acetate. Oxaloacetate is further broken down to pyruvate and CO 2 . Production of Na 2 CO 3 as well as NH 3 from utilization of sodium citrate and ammonium salt respectively results in alkaline pH. This results in change of medium’s color from green to blue.

57. Procedure: Simmon’s citrate agar media was prepared and sterilized using autoclave 5ml of media was poured into the culture tubes and slants were prepared Simmon’s citrate agar slants were inoculated with test organism The uninoculated tubes were kept as control The tubes were incubated at 37 C for 48hours Slant culture was observed for the growth and coloration of the medium

58. IMViC results Species Indole Methyl Red Voges-Proskauer Citrate Escherichia coli Positive Positive Negative Negative Shigella spp . Negative Positive Negative Negative Salmonella spp . Negative Positive Negative Positive Klebsiella spp. Negative Negative Positive Positive Proteus vulgaris   Positive Positive Negative Negative Proteus mirabils Negative Positive Negative Positive Citrobacter freundii Negative Positive Negative Positive Enterobacter aerogenes Negative Negative Positive Positive

59. Triple sugar iron agar(TSI) This test is used to determine the ability to: 1- Reduce sulfur into H2S. 2- Lactose & /or Sucrose fermentation. 3- Glucose fermentation and Gas production . Used to differentiate among the different groups of Enterobacteriaceae

60. Media Used: Triple sugar-iron agar TSI contains 10 parts lactose: 10 parts sucrose: 1 part glucose and peptone. Sodium thiosulfate & ferrous sulfate (iron). Phenol red and ferrous sulphate serves as indicators of acidification and H 2 S 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 buttom of the medium in the tube.

61. Procedure and result interpretation Inoculate TSI medium with an organism by inoculating needle by stabbing the butt & streaking the slant, incubate at 37°C for 24 hours. Alkaline slant/no change in the butt ( K/NC) = Glucose, lactose and sucrose non- utilizer (alkaline slant/alkaline butt) Alkaline slant/acid butt ( K/A) = Glucose fermentation only. Acid slant/acid butt ( A/A), with gas production = Glucose, sucrose , and/or lactose fermenter . Alkaline slant/acid butt ( K/A), H2S production = Glucose fermentation only.

62. SIM- SULFIDE,INDOLE, MOTILITY It is performed on sulfide- indole -motility (SIM) medium or in tryptophan broth, or in motility urease indole (MIU) medium . This is a differential medium. It tests the ability of an organism to do several things: reduce sulfur, produce indole and swim through the agar (be motile). SIM is commonly used to differentiate members of Enterobacteriaceae . If sulfide is produced, a black color forms in the medium. Proteus mirabilis is positive for H 2 S production .

63. Bacteria that have the enzyme tryptophanase , can convert the amino acid, tryptophane to indole . Indole reacts with added Kovac’s reagent to form rosindole dye which is red in color. Escherichia coli is indole positive. SIM tubes are inoculated with a single stab to the bottom of the tube. If an organism is motile then the growth will radiate from the stab mark and and make the entire tube appear turbid. Pseudomonas aeruginosa and Proteus mirabilis are motil e.