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![Gram stain
a. Gram stain divides the bacteria into Gram positive
& Gram negative.
The basic procedure :
i. Take a heat fixed bacterial smear.
ii. Flood the smear with CRYSTAL VIOLET or Methyl violet for 1
minute, then wash with water. [PRIMARY STAIN]
iii. Flood the smear with IODINE for 1 minute, then wash with water.
iv. Flood the smear with ETHANOL-ACETONE, quickly, then wash with
water. [DECOLORI
v. Flood the smear with SAFRANIN for 1 minute, then wash with
water. [COUNTERSTAIN]
vi. Blot the smear, air dry and observe.
Dr.T.V.Rao MD 12](https://image.slidesharecdn.com/identificationofbacteria-180120102225/75/Basics-in-Identification-of-Bacteria-by-Dr-T-V-Rao-MD-12-2048.jpg)
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Basics in Identification of Bacteria by Dr.T.V.Rao MD
The document discusses various methods for identifying bacteria, including microscopic examination of cell morphology and staining techniques like Gram staining, endospore staining, and flagella staining. It also covers biochemical tests like indole, methyl red, Voges-Proskauer, citrate, and hydrogen sulfide production that can help differentiate between bacterial species. The document provides examples of morphological characteristics and biochemical profiles that are used to identify common bacteria like Staphylococcus, Streptococcus, and members of the Enterobacteriaceae family.
In this document
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Introduction to the basics of bacterial identification by Dr.T.V.Rao. Highlights the importance and relevance of taxonomy.
Discussion on biological classification hierarchy: Family, Genus, Species, Type, and Strain.
Methods of bacterial identification including Microscopic Morphology, Serological Analysis, and Genetic Analysis.
Outlines steps for isolating bacteria from samples and identifying them once pure cultures are obtained.
Describes morphological characteristics of bacteria, focusing on shape types: cocci, bacilli, spiral.
Overview of the Gram stain technique, its steps and differentiation of gram-positive (purple) and gram-negative (pink) bacteria.
Gram stain morphology details including shapes, arrangements of bacteria, and their classification.
Focus on Gram-positive cocci such as Staphylococci and Streptococci, including characteristics and examples.
Details on Gram-negative cocci and bacilli, giving specific examples of harmful bacteria.
Contrast between Gram-positive and Gram-negative bacteria concerning their cell wall structure and implications.
Introduction to spirochetes and a dichotomous key for classification based on different characteristics.
Details on bacterial phenotypes, including isolation and characteristics of colony morphology.
Explains lactose fermentation testing, showing which bacteria can ferment lactose using MacConkey’s agar.
Overview of various biochemical tests such as Indole, MR, and VP for differentiating Enterobacteriaceae.
Details the principle and method of Indole test to identify certain bacterial species based on tryptophan metabolism.
Describes oxidase test procedures, quality controls, and reading results for testing cytochrome oxidase presence.
Reports on MR and VP tests for assessing glucose fermentation and acid production in Enterobacteriaceae.
Nitrate reduction test to differentiate bacteria based on their ability to reduce nitrate to nitrite or other products.
Tests for motility and urease activity to identify bacteria, detailing methods and expected results.
Explains the coagulase test for differentiating Staphylococcus aureus from other Staphylococci.
Overview of molecular analysis including DNA-DNA homology for assessing genetic relatedness among bacterial species.
Concluding remarks by Dr.T.V.Rao, encourages contact for further inquiries or collaborations.
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Basics in Identification of Bacteria by Dr.T.V.Rao MD
- 1. Basics in Identification ofBacteria Dr.T.V.Rao MD Dr.T.V.Rao MD 1
- 2. Q: What wasthat clue to help us remember the hierarchy of biological classification? From the Virtual Microbiology Classroom on ScienceProfOnline.com Dr.T.V.Rao MD 2
- 3. Classification Family: a groupof related genera. Genus: a group of related species. Species: a group of related strains. Type: sets of strain within a species (e.g. biotypes, serotypes). Strain: one line or a single isolate of a particular species. Dr.T.V.Rao MD 3
- 4. 4 Methods in bacterialidentification 1. Microscopic morphology - Gram Staining, Shapes, arrangements, motility 2. Macroscopic morphology – colony appearance, motility 3. Physiological / biochemical characteristics – aerobic, anaerobic, photosynthetic, growth on selective media 4. Chemical analysis – e.g. peptides and lipids in cell membranes Dr.T.V.Rao MD
- 5. Methods in bacterialidentification 1. Phage Typing – which phage infects the bacterium 2. Serological analysis – what antibodies are produced against the bacterium 3. Pathogenicity – what diseases does the bacterium cause. 4. Genetic & molecular analysis • G + C base composition • DNA analysis using genetic probes • Nucleic acid sequencing & rRNA analysis Dr.T.V.Rao MD 5
- 6. Steps in diagnosticisolation and identification of bacteria • Samples of body fluids are streaked on culture plates and isolated colonies of bacteria appear after incubation. Observation of these colonies for size, texture, color, and (if grown on blood agar) hemolysis reactions, is highly important as a first step in bacteria identification. Whether the organism requires oxygen for growth is another important differentiating characteristic. Dr.T.V.Rao MD 6
- 7. Identification of Bacteriaafter isolation in pure forms • The bacteria is obtained in pure culture it has to be identified Dr.T.V.Rao MD 7
- 8.
- 9. Morphology • Depends on numberof factors such as strain studied nature of the culture medium • Temperature and time of incubation Dr.T.V.Rao MD 9
- 10. Shape of thecolony Dr.T.V.Rao MD 10
- 11. Shape of Bacteria Bacteria display three basic shapes: i. round- cocci, (from the Greek kokkos - a berry), ii. rod shaped – bacilli (from the Latin bacillus - a stick or rod), iii.spiral (quelled).Dr.T.V.Rao MD 11
- 12. Gram stain a. Gramstain divides the bacteria into Gram positive & Gram negative. The basic procedure : i. Take a heat fixed bacterial smear. ii. Flood the smear with CRYSTAL VIOLET or Methyl violet for 1 minute, then wash with water. [PRIMARY STAIN] iii. Flood the smear with IODINE for 1 minute, then wash with water. iv. Flood the smear with ETHANOL-ACETONE, quickly, then wash with water. [DECOLORI v. Flood the smear with SAFRANIN for 1 minute, then wash with water. [COUNTERSTAIN] vi. Blot the smear, air dry and observe. Dr.T.V.Rao MD 12
- 13. Crystal violet Gram's iodine Decolorise with acetone Counterstain with e.g.methyl red Gram-positives appear purple Gram-negatives appear pink The Gram Stain
- 14.
- 15. Gram-positive rods Gram-negative rods Gram-positivecocci Gram-negative cocci
- 16.
- 17. Gram stain morphology •Shape – cocci (round) – bacilli (rods) – spiral or curved (e.g. spirochetes) • Single or multiple cells – clusters (e.g. streptococci) – chains (e.g. streptococci) • Gram positive or negative Dr.T.V.Rao MD 17
- 18. Gram-positive Cocci • Staphylococci –Catalase-positive – Gram-positive cocci in clusters • Staphylococcus aureus – coagulase-positive • Staph. epidermidis – and other coagulase negative staphylococci
- 19. Gram-Positive Cocci • Streptococci –Catalase-negative – Gram-positive cocci in chains or pairs • Strep. pyogenes • Strep. pneumoniae • Viridans-type streps • Enterococcus faecalis
- 20. Gram-Negative Cocci • Neisseriagonorrhoea – The Gonococcus • Neisseria meningitides – The Meningococcus • Both Gram-negative intracellular diplococci
- 21. Gram-Negative Bacilli • EntericBacteria – E. coli – Salmonella – Shigella – Yersinia – Pseudomonas – Proteus – Vibrio cholerae – Klebsiella pneumoniae
- 22. Classifying Bacteria: Gram-Negative& Gram-Positive Gram __________ • Peptidoglycan is the thick, outermost layer of the cell wall. • About 90% of cell wall is made of peptidoglycan. Gram __________ • Cell wall is more chemically complex, thinner and less compact. • Peptidoglycan only 5 – 20% of the cell wall. • Peptidoglycan is not the outermost layer, but between the plasma membrane and the outer membrane. • Not accessible to the action of antibiotics. • Outer membrane is similar to the plasma membrane, but is less permeable and contains lipopolysaccharides (LPS). • LPS is a harmful substance classified as an endotoxin. From the Virtual Microbiology Classroom on ScienceProfOnline.com Dr.T.V.Rao MD 22
- 23. Spirochetes • Thin spiralbacteria • Viewable by phase- contrast microscopy or silver stain – Trepanoma palladium – Borrelia burgdorferi – Leptospira
- 24. Classification: Dichotomous Key SimpleStain Cocci Bacilli Gram Stain Gram negative cocci Gram positive cocci Mannitol Salt yellow pink Staphylococcus aureus Staphylococcus epidermis Gram Stain Gram negative bacilli Gram positive bacilli MacConkey’s No color change Salmonella pullorum Pink colonies E. coli Enterobacter aerogenes Acid Fast stain Acid Fast Mycobacterium tuberculosis Not acid fast Endospore stain Forms endospores Bacillus subtilus smegmatis Since we will be working with a limited number of bacterial species and identification techniques, we will be using a limited dichotemous key in lab. Dr.T.V.Rao MD 24
- 25. GRAM-POSITIVE Facultative anaerobe, cocci Thick cell wall, ~50% of cell’s mass. (When you Gram stain it, the cells are intensely purple.) Found in many places throughout the environment human skin, animals, water, dust, and soil. M. luteus on human skin transforms chemicals in sweat into body odor. Grow well even with little water or high salt concentrations. (You may find it growing on your Mannitol Salt nasal sample.) Normal flora that can become opportunistic in immune compromised. Bacterial Genus: _______________ This is your lab friend Micrococcus luteus. Images: M. luteus colonies, T.Port; M. luteus, Janice Carr, PHIL #9761 Dr.T.V.Rao MD 25
- 26. Isolation in pureform Studies on the biochemical, antigenic and other characters of bacteria can be done only if the organism available in the pure form. Technique: a. Plating on solid culture media- clinical sample is streaked onto a solid medium (like: MacConkey agar, nutrient agar or blood agar) in such a way so as to ensure isolated discrete colonies. b. Use of selective growth condition-most important example of this is the growth of anaerobic bacteria which will not take place in an environment having oxygen. Dr.T.V.Rao MD 26
- 27. Isolation of Bacteriaon Culture plates Dr.T.V.Rao MD 27
- 28. Dr.T.V.Rao MD Flagella • Motility- movement • Swarming occurs with some bacteria – Spread across Petri Dish – Proteus species most evident • Arrangement basis for classification – Monotrichous; 1 flagella – Lophotrichous; tuft at one end – Amphitrichous; both ends – Peritrichous; all around bacteria. 28
- 29.
- 30. India ink (capsulestain) • The capsule stain employs an acidic stain and a basic 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. Dr.T.V.Rao MD 30
- 31.
- 32. Different types ofHaemolysis Dr.T.V.Rao MD 32
- 33. Different types ofHaemolysis Dr.T.V.Rao MD 33
- 34. On solid mediumthe following characters are observed i. Shape: circular, irregular, radiate or rhizoid. ii. Size: The size of the colony can be a useful characteristic for identification. The diameter of a representative colony may be measured. iii. Elevation: iv. Margin: Entire, wavy, lobate, filiform v. Surface: smooth, wavy, rough, granular, papillate, glistening etc. vi. Size in mm vii. Texture : dry, moist, mucoid, brittle, viscous, butyrous (buttery). viii. Color : colorless, pink, black, red, bluish-green.Dr.T.V.Rao MD 34
- 35. contd Bacillus subtilis Proteusspp.Dr.T.V.Rao MD 35
- 36. Lactose Fermentation MacConkey’s agarhelps us identify different types of Gram negative bacteria. There is lactose in the medium that the bacteria can eat, and a pH sensitive dye that turns pink, if they “poop out” waste from breaking down lactose. Lac+ genera include E. coli, Enterobacter, and Klebsiella. Lac- genera include Salmonella, Shigella, & Proteus. Image: MacConkey’s agar, T. Port Dr.T.V.Rao MD 36
- 37. Tests To Know •Case Study Tests – Indole – Methyl Red/Voges Proskauer – Citrate – H2S production in SIM – Motility – Lactose fermentation – Sucrose fermentation – Glucose fermentation & gas production – Triple Sugar Iron Agar (TSI) test • Staphylococcus identification tests – MSADr.T.V.Rao MD 37
- 38. PHENOTYPIC CHARACTERISTICS Metabolic differences •Biochemical tests –Sugar fermentation • e.g., Lactose, sucrose, glucose, etc. • Fermentation results in acid production – pH indicator changes color – Pink yellow • Inverted tube (Durham tube) collects any gas produced
- 39. Key identification characteristicsfor Enterobacteriaceae GENUS/SPECIES Fermentation of Gas MR VP Indole Citrate Urease H2S M G L S M Escherichia coli (+) (+) (+) (+) (+) (+) (-) (+) (-) (-) (-) Shiegella (+) (-) (-) (+) (-) (+) (-) (-/+) (-) (-) (-) Shiegella sonnei (+) (+) (-) (+) (-) (+) (-) (-) (-) (-) (-) Salmonella (+) (-) (-) (+) (+) (+) (-) (-) (+) (-) (+) Klebsiella Pneumo. (+) (+) (-) (+) (+) (-) (+) (-) (+) (+) (-) Enterobacter (+) (-) (+) (+) (-) (+) (-) (+) (-) (+) (+) Serratia (+) (+) (-) (+) (+) (-/+) (+) (-) (+) (-) (-) Proteus (+) (-) (-) (+) (-/+) (+) (-) (+) (-/+) (+) (+) morganella (+) (-) (-) (+) (+) (+) (-) (+) (-) (+) (+) Yersinia (+) (-) (-) (+) (-) (+) (-) (-/+) (-) (-/+) (-) G: Glucose, L:Lactose, S:Sucrose, M: Manitol, MR: Methyl Red, VP: Voges Proskauer Dr.T.V.Rao MD 39
- 40. Indole Test Principle: Indole testis 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. Dr.T.V.Rao MD 40
- 41. Indole Test Property ittests 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. Dr.T.V.Rao MD 41
- 42. Indole test • Procedure: -InoculateTryptone 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 aerogensDr.T.V.Rao MD 42
- 43. Oxidase Test Principle Oxidase testis 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.as positive test. No colour development indicates a negative test and the absence of the enzyme. Dr.T.V.Rao MD 43
- 44. Oxidase Test…. Methods Quality controls Positivecontrol- Pseudomonas spp Negative control – E. coli 1. Moist filter paper method 2. Direct plate method . 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 Dr.T.V.Rao MD 44
- 45. Oxidase Test….. Positive • Pseudomonasspp. • Aeromonas spp. • Vibrio spp. • Alcaligenes spp. • Neisseria spp. • Haemophilus sps Negative • Enterobacteriaceae • Acenitobacter spp. Dr.T.V.Rao MD 45
- 46. Methyl 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. Dr.T.V.Rao MD 46
- 47. Principle of MRtest: 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. Dr.T.V.Rao MD 47
- 48. 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) Left: negative/Right: positive MR Positive: E. coli MR Negative: Enterobacter aerogenes Enterobacter cloacae Klebsiella spp. Dr.T.V.Rao MD 48
- 49. Nitrate reduction…. • Todistinguish 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. Dr.T.V.Rao MD 49
- 50. Negative Negative Posiitive Positive Nitrate reducedto NH3 or N2 gas, nitrite absent Nitrate reduced to nitrite Nitrate not reducedNitrate not reduced Nitrate reduction test…. Dr.T.V.Rao MD 50
- 51. Addition of Zndust or Nitrate reduction test…. Dr.T.V.Rao MD 51
- 52. 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) Left: negative/Right: positive MR Positive: E. coli MR Negative: Enterobacter aerogenes Enterobacter cloacae Klebsiella spp. Dr.T.V.Rao MD 52
- 53. Motility Test • Propertyit 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. Dr.T.V.Rao MD 53
- 54. Urea Hydrolysis (Ureasetest) • 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. Dr.T.V.Rao MD 54
- 55. Reading Results: • Ureabroth 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. Dr.T.V.Rao MD 55
- 56. Coagulase test Principle: - Thistest 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 fibrinogen. When a bacterial suspension is mixed with plasma, this enzyme causes alteration in fibrinogen of the plasma to precipitate on the staphylococcal cells, causing the cells to clump. Dr.T.V.Rao MD 56
- 57. Coagulase test • Freecoagulase is produced extra- cellularly by the bacteria that causes the formation of a clot when S. aureus colonies are incubated with plasma Dr.T.V.Rao MD 57
- 58. Coagulase Results ReadingResults: 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 confirmed using the tube test. B. Tube test: - Positive: Clot of any size (a) - Negative: No clot (b) a b Coagulase Positive : Staphylococcus aureus Coagulase negative: Staphylococcus epidermidisDr.T.V.Rao MD 58
- 59. Molecular analysis It wouldbe ideal to compare sequences of entire bacterial chromosomal DNA. Alternatively, genomic similarity has been assessed by the guanine (G)+ cytosine (C) content (% GC). This has been replaced by two alternatives: 1. Hybridization 2. Sequencing specific genes Dr.T.V.Rao MD 59
- 60. DNA-DNA homology 1. Howwell two strands of DNA from different bacteria bind (hybridize) together. This technique is employed to compare the genetic relatedness of bacterial strains/species. 2. If the DNA from two bacterial strains display a high degree of homology (i.e. they bind well) the strains are considered to be members of the same species. Dr.T.V.Rao MD 60
- 61. • Program createdby Dr.T.V.Rao MD for benefit of Medical Microbiologists • Email • [email protected] Dr.T.V.Rao MD 61