Identification of bacteria using staining techniques

Identification of bacteria using staining techniques and Biochemical test IMViC test

1. UNIT II 2.1 Identification of bacteria using staining techniques and IMViC biochemical test Presented by: Mohammad Abuzar( M. Pharm ) Assistant Professor School of Pharmacy AIKTC, New Panvel .

2. CONTENTS 2

3. 3 INTRODUCTION Staining simply means colouring the microorganisms with a dye that emphasizes certain structures Need for staining Most microorganisms appear almost colourless when viewed through a standard light microscope Hence must be fixed and stained to Increase visibility Accentuate specific morphological features Preserve them for future study

4. 4 Types of Staining Simple Staining Differential Staining Special Staining Negative Staining Positive Staining Gram Staining Acid Fast Staining Capsule Staining Flagella Staining Endospore Staining

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6. 6 Basic staining procedure   Step 1: Smear preparation A thin film of material containing the microorganisms is spread over the surface of the slide. This film, called a smear. It is allowed to air dry. Step 2: Fixation By passing it through the flame of a bunsen burner several times, smear side up, or by covering the slide with methyl alcohol for one minute. Fixing simultaneously kills the microorganisms and fixes them to the slide. It also preserves various parts of microbes in their natural state with only minimal distortion Step 3: Staining Stain is applied and then washed off with water Slide is blotted with absorbent paper. The stained microorganisms are now ready for microscopic examination.

7. 1) Smear Preparation 3) Staining 2) Smear Fixation

8. 8 Stains Stains are salts composed of a positive and a negative ion, The colored ion is known as the chromophore. Stain Positive ion, Colored chromophore : Basic stain Negative ion, Colored chromophore: Acidic stain Bacteria are slightly negatively charged at pH 7. Thus, the colored positive ion in a basic dye is attracted to the negatively charged bacterial cell. Basic dyes include crystal violet, methylene blue, malachite green and safranin Acidic dyes are not attracted to most types of bacteria The dye's negative ions are repelled by the negatively charged bacterial surface The dye colors the background instead Preparing colorless bacteria against a colored background is called negative staining . Examples of acidic dyes are eosin, acid fuchsin, and nigrosine.

9. 9 Negative staining Valuable for observing overall cell shapes, sizes, and capsules The cells are made highly visible against a contrasting dark background Distortions of cell size and shape are minimized because fixing is not necessary and the cells do not pick up the stain

10. 10 Simple staining

11. A simple stain is an aqueous or alcohol solution of a single basic dye The primary purpose of a simple stain is to highlight the entire microorganism so that cellular shapes and basic structures are visible The stain is applied to the fixed smear for a certain length of lime and then washed off The slide is dried and examined Simple stains commonly used in the laboratory - methylene blue, carbolfuchsin, crystal violet, and safranin. Mordant Mordant - A chemical that intensifies the stain Functions of mordant To increase the affinity of a stain for a biological specimen To coat a structure (such as a flagellum) to make it thicker and easier to see after it is stained with a dye. 11

12. 12 Differential stains Differential stains react differently with different kinds of bacteria Can be used to distinguish them The differential stains most frequently used for bacteria are the Gram stain and the acid-fast stain Gram stain Gram stain was developed in 1884 by the Danish bacteriologist Hans Christian Gram Most useful staining procedures - classifies bacteria into two large groups: gram-positive and gram-negative. Step 1: Primary stain Step 2: Mordant Step 3: Decolorization Step 4: Counterstain

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14. 14 The purple dye and the iodine combine in the cytoplasm of each bacterium and color it dark violet or purple. Bacteria that retain this color after the alcohol has attempted to decolorize them are classified as gram-positive Because gram-positive bacteria retain the original purple stain, they are not affected by the safranin counterstain Bacteria that lose the dark violet or purple color after decolorization are classified as gram negative Because gram-negative bacteria are colorless after the alcohol wash, they are no longer visible. This is why the basic dye safranin is applied; it turns the gram-negative bacteria pink. Stains such as safranin that have a contrasting color to the primary stain are called counterstains

15. 15 Principle of Gram stain Different kinds of bacteria react differently to the Gram stain Structural differences in their cell walls affect the retention or escape of a combination of crystal violet and iodine, called the crystal violet- iodine (CV-I) complex Gram-positive bacteria have a thicker peptidoglycan cell wall than gram-negative bacteria Gram- negative bacteria contain a layer of lipopolysaccharide (lipids and polysaccharides) as part of their cell wall Crystal violet + Iodine Enters Gram positive cell wall à Peptidoglycan layer retains CV-I during alcohol decolorization à Gram- positive cells retain the color of the crystal violet dye Enters Gram negative cell wall à Alcohol wash disrupts the outer lipopolysaccharide layer à CV- I complex is washed ou tthrough the thin layer of peptidoglycan à Gram negative Cells are colorless à Turn pink upon safranin staining

16. 16 Clinical significance of Gram staining Gram reaction of a bacterium can provide valuable information for the treatment of disease. Gram-positive bacteria tend to be killed easily by penicillins and cephalosporins. Gram-negative bacteria are generally more resistant because the antibiotics cannot penetrate the lipopolysaccharide layer.

17. 17 Acid-Fast Stain or Ziehl- Neelsen Technique-AFB Staining Used to identify all bacteria in the genus Mycobacterium and Nocardia Mycobacterium tuberculosis, the causative agent of tuberculosis Mycobacterium leprae the causative agent of leprosy Acid-fast stain binds strongly only to bacteria that have a waxy material in their cell walls Cell wall of acid fast bacteria

18. 18 Acid fast staining Procedure The red dye C arbol fuchsin is applied to a fixed smear The slide is gently heated for several minutes((Heating enhances penetration and retention of the dye). The slide is cooled and washed with water. Smear is next treated with acid-alcohol, a decolorizer, which removes the red stain from bacteria that are not acid -fast. The acid-fast microorganisms retain the red color because the carbol fuchsin is more soluble in the cell wall lipids than in the acid-alcohol The smear is then stained with a methylene blue counterstain. Non acid-fast cells appear blue after application of the counterstain

19. 19 Special stains Special stains are used to color and isolate specific parts of microorganisms, such as endospores and flagella, and to reveal the presence of capsules. 1. Negative Staining for Capsules 2. Endospore (Spore) Staining 3. Flagella Staining B. Subtilis Endospore (Spore) Staining Negative Staining for Capsules Flagella Staining

20. 20 1)Negative Staining for Capsules Many microorganisms contain a gelatinous covering called a capsule Demonstrating the presence of a capsule is a means of determining the organism's virulence, the degree to which a pathogen can cause disease. Step 1 : Mix the bacteria in a solution containing a fine colloidal suspension of colored particles (usually India ink or nigrosin ) Step 2 : Provide a contrasting background and then stain the bacteria with a simple stain, such as safranin Due to their chemical composition, capsules do not accept most biological dyes, such as safranin, and thus appear as halos surrounding each stained bacterial cell.

21. 21 2) Endospore (Spore) Staining Endospore is a special resistant, dormant structure formed within a cell Protects a bacterium from adverse environmental conditions Endospores cannot be stained by ordinary methods The dyes do not penetrate the wall of the endospore Schaeffer- Fulton endospore stain Malachite green, the primary stain, is applied to a heat-fixed smear Heated to steaming for about 5 minutes ( i.e , malachite green permeate the spore wall ) Washed for about 30 seconds with water to remove the malachite green from all of the cells parts except the endospores. Safranin, a counterstain, is applied to the smear to stain portions of the cell other than endospores. The endospores appear green within red or pink cells

22. 22 Note : In Gram Staining and AFB Staining we use Alcohol or Acid Alcohol or Acid as a decolorizer but in spore staining water is sufficient ( to be used as decolorizer) because: malachite green dye is water-soluble and does not adhere well to the cell wall vegetative cells have been disrupted by heat, because of these reasons, the malachite green rinses easily from the vegetative cells 3) Flagella staining Bacterial flagella (singular: flagellum) are structures of locomotion too small to be seen with a light microscope without staining. A tedious and delicate staining procedure uses a mordant and the stain carbol fuchsin to build up the diameters of the flagella until they become visible under the light microscope

23. 23 IMViC Test (Biochemical test) The IMViC test is a series of four different biochemical tests used in identifying and differentiating bacteria, especially the members of  Enterobacteriaceae . Though it can be (and is) used for the identification of any type of bacteria, it is mainly used for identifying Gram-negative bacteria. 

24. 24 IMViC is an acronym for four different biochemical tests; each letter except “I” represents an individual test making this series of biochemical tests. IMViC series contains the following biochemical tests: “I” = Indole Test “M” = Methyl Red (MR) Test “V” = Voges – Proskauer (VP) Test “C” = Citrate Utilization Test (simply Citrate Test) The letter  “ i ”  after ‘V’ is only for the rhyming purpose, it does not indicate any test.   On making a minor modification like the use of  Sulfide-Indole-Motility (SIM)  Agar medium, instead of 4, we can test 6 biochemical properties. The use of SIM medium gives information on the ability to test bacteria’s ‘motility’ and ‘sulfide production’ (H 2 S production) ability. Hence, it is a widely accepted series of biochemical tests.

25. Objectives of the IMViC Test To study some biochemical properties – indole production, acid production, acetylmethylcarbinol (acetoin) production, and citrate utilization – of isolated unknown bacteria in order to characterize and identify them. To selectively differentiate and identify members of the  Enterobacteriaceae  family. Principle of IMViC Test The IMViC test is based on the variations in the metabolic requirements and properties of different genera and species of bacteria.  The ‘indole test’ and ‘citrate utilization test’ in the series detect the ability of bacteria to produce specific enzymes and utilize specific nutrients. On the other hand, the ‘MR test’ and ‘VP test’ in the series detect the final metabolic products produced by the bacteria utilizing specific nutrients. 25

26. 26 What is Indole Test? The indole test  is a biochemical test in the IMViC test series which detects the ability of organisms (bacteria) to produce indole as a metabolic product utilizing tryptophan. It is indicated by the letter “I” of the IMViC . Principle of Indole Test Some bacteria can produce an enzyme called ‘tryptophanase’ which helps them to metabolize the amino acid ‘tryptophan’ into ‘indole, pyruvic acid, and ammonia’. When the indole reagent is added to a medium with a bacterial culture that has produced indole, the indole combines with the aldehyde present in the reagent to give a distinctive color.  If benzaldehyde is present in the reagent, a pink to a violet-red quinoidal compound is formed, hence; a pink to red color ring is formed.  If cinnamaldehyde is present in the reagent, a blue to the green color compound is formed, hence; a green to blue color ring is formed. 

27. 27 Test bacteria are cultured in the medium containing tryptophan for 24 – 48 hours and an indole reagent is added following the incubation to read the result. A positive result is indicated by the formation of a pink to violet-red or green to blue color ring according to the type of reagent used. e.g. Escherichia coli, Klebsiella oxytoca , V. cholerae, Proteus vulgaris, Porphyromonas asaccharolytica , Vibrio spp., A negative color is indicated by the formation of a lack of color change or a slight yellowish color ring at the top.  e.g. Klebsiella pneumoniae, Proteus mirabilis, Salmonella spp., Shigella spp., Citrobacter freundii , Pseudomonas aeruginosa,

28. 28 What is Methyl Red (MR) Test? Methyl Red (MR) Test  is a biochemical test that detects the ability of organisms (bacteria) to produce stable mixed acids as metabolic end products of glucose metabolism. It is indicated by the letter “M” of the IMViC . Principle of MR Test Some species of bacteria use the mixed acid fermentation pathway as their glucose metabolism process. Following this metabolic pathway, they convert pyruvate into stable mixed acids. When such acid fermenters bacteria are grown in a medium containing glucose (or carbohydrate), they will release the acids, hence; decreasing the pH of the medium to 4.4 or lower. When methyl red indicator is added in a medium containing such acid fermenters, it will turn the medium red.”

29. 29 Following the 24-hour incubation on the MR-VP broth, a methyl red indicator is added to the broth. A positive result is indicated by the development of red color while a negative result is indicated by the development of yellowish color. MR Positive Bacteria :  Escherichia coli, Salmonella spp., Shigella spp., Citrobacter spp., Proteus spp., Yersinia spp., Edwardsiella spp., Staphylococcus aureus,  MR Negative Bacteria :  Klebsiella pneumoniae, Enterobacter spp., Hafnia spp., Serratia marcescens .

30. What is Voges-Proskauer (VP) Test? Voges-Proskauer (VP) Test  is a biochemical test in the IMViC test series which detects the ability of organisms (bacteria) to metabolize the pyruvate into a neutral intermediate product called ‘acetylmethylcarbinol’ or ‘acetoin’. It is indicated by the letter “V” of the IMViC . Principle of VP Test Pyruvate can be metabolized into a neutral intermediate product called ‘acetyl methyl carbinol’, commonly called the ‘acetoin’ during the butanediol pathway of 2,3-butanediol production. 30

31. Following the 48-hour aerobic incubation on MR-VP broth, VP reagents I and II are added and the color change is observed within 30 minutes. A positive result is indicated by the development of pink – red color at the top of the broth immediately or within 30 minutes but not more than 1 hour. e.g. Klebsiella spp., Enterobacter spp., Viridans Streptococci (except S. mitis, and S. vestibularis ), Proteus mirabilis, Hafnia spp., Serratia spp. No change in color represents a negative VP test. e.g. Escherichia spp.,   Proteus vulgaris, Citrobacter freundii ,  31

32. What is Citrate Utilization Test? Citrate Utilization Test  is a biochemical test in the IMViC test series which detects the ability of organisms (bacteria) to utilize citrate as a sole source of energy. It is indicated by the letter “C” of the IMViC .  Principle of Citrate Utilization Test Some bacteria can utilize ‘citrate’ as their sole source of carbon. Such bacteria produce citrase enzymes which will break the citrate into oxaloacetic acid and acetic acid. The oxaloacetic acid will then be decarboxylated to produce pyruvate and CO 2 . Released CO 2  will combine with H 2 O and excess sodium from sodium citrate to produce alkaline ‘sodium carbonate’.  The sodium carbonate will increase the pH of the medium. 32

33. CO 2  + H 2 O + excess sodium from sodium citrate → Na 2 CO 3  (alkaline) Additionally, the released CO 2  will trigger the metabolism of ammonium salts. Utilization of the ammonium salts as a source of nitrogen will cause the production of ammonia (or ammonium hydroxide).  Ammonium salt → Ammonium hydroxide (alkaline)  The combined effect of ammonium hydroxide and sodium carbonate will increase the pH of the media above 7.6. This increase in pH will turn the pH indicator bromothymol blue in the medium from deep forest green (at neutral pH) to Prussian blue. 33

34. 34 Following the incubation of 24 – 48 hours (up to 4 days for some), bacterial growth and color change in the slant portion is observed. A positive result is indicated by growth and change in color of slant from green to intense blue. A negative result is indicated by no change in the color of the slant. Citrate Positive Bacteria : Klebsiella spp., Citrobacter spp., Serratia marcescens, Proteus mirabilis, Enterobacter spp., Salmonella spp. (except Salmonella Typhi and Paratyphi A. Citrate Variable Bacteria : Proteus vulgaris, V. cholera, V. parahaemolyticus. Citrate Negative Bacteria : Escherichia coli, Shigella spp., Salmonella Typhi and Paratyphi A, Yersinia spp., Morganella morganii, Staphylococcus aureus, etc.

35. Uses of IMViC Test It is routinely used in clinical, research, and teaching laboratory to characterize and identify unknown isolated bacteria up to the level of genus.  Alongside the Urease test and TSI (Triple Sugar Iron) test, the test can differentiate and identify the members of the  Enterobacteriaceae  family. Some tests are used to differentiate species of a genus. E.g., the indole test is used to differentiate  K. oxytoca   and  K. pneumoniae, C. koseri   and  C. freundii , P. vulgaris  and  P. mirabilis , etc.   35

36. Limitations of IMViC Test It is not enough to completely identify the bacteria up to the level of species. Additional tests are required, even for differentiating Enterobacterales .  Different genera give the same results, hence results become ambiguous.  It is culture-based test series and hence requires a longer period. Some bacteria require more than 2 days of incubation to show VP reaction, and for the citrate test, we may have to incubate for more than 4 days.  Only the culturable bacteria can be characterized.  The whole process is complex and requires good culture skills and various resources to do the procedure.  False positive results and false negative results are encountered if the incubation period is not appropriate and the reagents are old or not used appropriately. 36

37. 37 Summary Morphological stains help in identifying the cell size, shape and structure Simple stains color the cells Negative staining color the background Gram staining differentiates between gram positive and gram negative cells The difference in gram staining is due to difference in cell wall composition Stages of gram staining – Primary stain Mordant Decolorization Counterstaining Acid fast staining is used to identify bacteria containing mycolic acid in cell wall – belonging to genus Mycobacterium and Nocardia It differentiates between acid fast and non-acid fast organisms Special structural stains like capsule spore and flagella staining, help in visualization of these bacterial structures

38. 38 W.B. Hugo and A.D. Russel: Pharmaceutical Microbiology, Blackwell Scientific publications, Oxford London. Prescott and Dunn., Industrial Microbiology, 4th edition, CBS Publishers & Distributors, Delhi. Pelczar , Chan Kreig , Microbiology, Tata McGraw Hill edn . Malcolm Harris, Balliere Tindall and Cox: Pharmaceutical Microbiology. Rose: Industrial Microbiology. Probisher , Hinsdill et al: Fundamentals of Microbiology, 9th ed. Japan Cooper and Gunn’s: Tutorial Pharmacy, CBS Publisher and Distribution. Peppler : Microbial Technology. I.P., B.P., U.S.P.- latest editions. Ananthnarayan : Text Book of Microbiology, Orient-Longman, Chennai Edward: Fundamentals of Microbiology. 12. N.K.Jain : Pharmaceutical Microbiology, Vallabh Prakashan , Delhi REFERENCES

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