General Characteristics of microbes.pptx

General Characteristics of Microbes is the 2nd unit of Microbiology subject for Nursing students. Students are able to understand the classification, morphology, structure, growth & nutrition, laboratory methods for the identification of bacteria.

1. GENERAL CHARACTERISTICS OF MICROBES UNIT 2

2. Classification of Microorganisms

3. Classification - orderly arrangement of units under study into groups of larger units. Carolus Linnaeus (1707-1778) - Established classification in Biology. Microorganisms are classified according to 9 different characteristics.

4. Classification on the basis of ;- Body & nuclear organisation Morphological characteristics Nutritional requirements Oxygen requirements Temperature requirements Osmotic condition requirements Distribution Economical importance Host resistance or Pathogenicity

5. Classification on the Basis of Body & Nuclear Organisation

6. On the basis of body organisation:- Microbes beyond cellular organisation - These organisms completely lack cell structure in their body. Eg :- Viruses Microbes with cellular organisation - these organisms have their body made up of cells. Eg:- Bacteria, microalgae, microfungi, protozoans etc.

7. On the basis of nuclear organisation :- Prokaryotes - Microorganisms that possess incipient nucleus & lacks nuclear membrane, nucleoplasm & nucleolus. Eg:- Archaebacteria, Cyanobacteria & Eubacteria Eukaryotes - Microorganisms that possess a well developed nucleus with nuclear membrane, nucleoplasm & nucleolus. Eg:- Microalgae, protozoans, slime moulds, microfungi

8. 2. Classification on the Basis of Morphological Characteristics

9. I According to Shape of Bacteria COCCI (Spherical or Oval) Cocci in cluster. Eg:- Staphylococcus Cocci in chain Eg:- Streptococci Cocci in pair Eg:- Diplococcus pneumonia Cocci in groups of four -tetrad Eg:- Gaffkya tetragena Cocci in groups of eight Eg:- Sarcina lutea

10. Staphylococcus Streptococcus

11. Diplococcus pneumonia Gaffkya tetragena Sarcina lutea

12. 2. BACILLUS (Cylindrical or Rod shaped) Coccobacilli :- length of the cell greater than its breadth. Eg:- Brucella Cuneiform. Eg:- Corynebacterium Vibrio:- comma shaped, curved rods. Eg:-Vibrio cholerae Spirilla :- Rigid spiral shaped bacteria Spirochetes :- longer, thinner, flexible & coil shaped bacteria Actinomycetes :- branching type filamentous bacteria

13. COCCOBACILLI CUNEIFORM VIBRIO SPIRILLA SPIROCHETES ACTINOMYCETES

14. 3. Bacteria without stable shape Lacks cell wall Does not have stable morphology Called as MYCOPLASMA They occur as round & oval bodies & as interlacing filaments

15. II According to their Size Higher bacteria :- they are branching filamentous bacteria. Eg:- Actinomycetes Lower bacteria :- they are single, small prokaryotic cells. Eg:- Cocci, Bacilli, Vibrio, Spirochetes etc.

16. III According to their Flagella Monotrichous :- bacteria with single polar flagellum. Eg:- Vibrio cholerae Amphitrichous :- bacteria with single polar flagella or tuft of flagella at both poles. Eg:- Alcaligenes faecalis Lophotrichous :- Bacteria with tuft of flagella at one pole. Eg:- Spirilla Peritrichous :- Bacteria with flagella all around the cell. Eg:- Salmonella typhi.

17. IV According to their Staining Reaction GRAM STAIN GRAM POSITIVE BACTERIA:- The bacteria which appear violet after gram staining. Eg:- Staphylococcus aureus GRAM NEGATIVE BACTERIA :- The bacteria which appear pink or red after gram staining. Eg:- E.coli ) ACID-FAST or ZIEHL-NEELSEN STAIN ACID FAST ORGANISM :- The bacteria which appear pink in colour after staining. Eg:- Mycobacterium group NON ACID FAST ORGANISM :- The bacteria which appear blue in colour after staining. Eg:- E.coli

18. A) Non Acid Fast B) Acid Fast

19. V Based on Biochemical Reaction Basic classification:- AEROBIC & FACULTATIVE AEROBIC BACTERIA ANAEROBIC BACTERIA Aerobic & facultative aerobic bacteria are further classified into:- Cocci : Gram Positive Catalase positive : Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus Catalase negative : Staphylococcus pyogenes, Staphylococcus pneumoniae, Viridans group Streptococci Cocci : Gram Negative :- Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis

20. Bacilli : Gram Positive : Bacillus anthracis, Bacillus cereus, Corynebacterium , Mycobacterium Bacilli : Gram Negative Enterobacteriaceae :- E.coli, Klebsiella, Salmonella, Shigella Non- Enterobacteriaceae - Fermentive Bacilli :- Aeromonas, Vibrio, Pasteurella Non-Enterobacteriaceae -Non Fermentive Bacilli :- Pseudomonas, Moraxella, Acineobacter, Eikenella Coccobacilli : Gram Negative :- Actinobacillus, Bartonella, Brucella, Haemophilus, Chlamydia

21. Mycoplasma :- Mycoplasma hominis, Mycoplasma pneumonia, Ureaplasma urealyticum Treponema :- Treponema pallidium, Leptospira, Borelia burgdorferi, Borelia recurrentis

22. 3. Classification on the basis of Nutritional Requirements

23. PHOTOAUTOTROPHS :- Use light energy to produce food. Eg:- photosynthetic microalgae, cyanobacteria CHEMOAUTOTROPHS :- Use chemical energy to produce food. Eg:- sulphur bacteria, iron bacteria, nitrifying bacteria PHOTOHETEROTROPHS :- These are microbes that use light energy in obtainment of their food from external environment. Eg:- purple non-sulphur bacteria CHEMOHETEROTROPHS :- These are microbes that use chemical energy in obtainment of their food from external environment. Eg:- Saprophytic bacteria, symbiotic bacteria, microfungi, protozoa, colorless microalgae.

24. 4 . Classification on the basis of Oxygen Requirements

25. OBLIGATE / TRUE AEROBES :- Grow in presence of Oxygen only. OBLIGATE / TRUE ANAEROBES :- Grow in absence of Oxygen only. FACULTATIVE AEROBES :- Grow even in absence of Oxygen. FACULTATIVE ANAEROBES :- Grow even in presence of Oxygen.

26. 5. Classification on the basis of Temperature Requirements

27. PSYCHROPHILIC / CRYOPHILIC :- Grow at sufficiently low temperature. Best growth occurs at 0 deg as the minimum, 10-15 deg as optimum & below 20 deg as maximum. MESOPHILIC :- Grow on moderate temperature. Best growth occurs at 10-15 deg as the minimum, 30-40 deg as optimum & below 45 deg as maximum. THERMOPHILIC :- Grow at higher a temperature lethal to many other microbes . Best growth occurs at 45 deg as the minimum, 50-85 deg as optimum & above 100 deg as maximum.

28. 6. Classification on the basis of Osmotic Condition Requirements

29. OSMOPHOBIC :- These microbes die if subjected to substrate of high osmotic concentrations. OSMOPHILIC :- These microbes grow best on substrates of high osmotic concentrations./ HALOPHILIC :- These microbes preferably grow in high osmotic concentrations produced by dissolved salts. OSMODURIC :- These microbes grow normally on substrates of moderate osmotic concentrations & are resistant to wide osmotic changes in the substrate on which they\ grow.

30. 7. Classification on the basis of Distribution

31. HYDROSPHERIC / AQUATIC :- These microbes grow in water. LITHOSPHERIC / TERRESTRIAL :- These microbes grow on or in soil or rocky substances. ATMOSPHERIC / AERIAL :-These microbes are found in atmosphere.

32. 8. Classification on the basis of Economical Importance

33. USEFUL MICROBES HARMFUL MICROBES

34. 9. Classification on the basis of Host Resistance or Pathogenicity

35. PATHOGENS :- These microbes causes disease in the host. Eg:- Vibrio cholerae NON-PATHOGENS :- These microbes do not cause any disease in the host. Eg:- Lactobacilli.

36. B) Morphology of Bacteria

37. SIZE OF BACTERIA Size of bacterial cell is 1/ 10th size of eukaryotic cells. 0.5-5.0 micrometers in length Size of medically important bacteria :- 0.2-1.5 micrometers in diameter & 3-5 micrometers in length Smallest bacteria :- Mycoplasma (0.3 micrometers)

38. SHAPE OF BACTERIA / MORPHOLOGICAL TYPES COCCI Micrococci Diplococci Streptococci Staphylococci Tetrad Sarcina (Octad) 2. BACILLI Micro Bacilli Diplobacilli Streptobacilli Palisade arrangement (bacilli lined side by side like matchsticks & at angles to one another. Eg:- Corynebacterium) diphtheria Comma shaped Spirillum - bacilli

39. Corynebacterium diphtheria

40. 3. COCCOBACILLI 4. SPIROCHETES

41. STRUCTURE OF BACTERIA

42. STRUCTURE OF A TYPICAL BACTERIAL CELL

43. Structures External to Cell Wall FLAGELLA They are long, hollow, helical filamentous organs of locomotion. Arise from cytoplasmic membrane & pass out through cell wall. Size:- 10-20 nm in diameter & 3-20 micrometer in length. Found in both gram positive & negative bacteria.

44. Structure of Flagellum

45. 3 Parts of flagellum are :- Basal body :- It represents a border & thicker basal region of flagellum attached with plasma membrane. Hook : - It represents a border & thicker basal region of flagellum & passes out through the cell wall. Filament :- It is the thinner, elongated & terminal part of the flagellum. The basal body comprises of a rod & two or more sets of encircling innermost (M & S) & outer most (P & L) rings. Gram negative bacteria consists of all these 4 rings.

46. Flagella is attached to cell membrane through M ring. Ring S is located just above the cell membrane. Flagella is attached to peptidoglycan & outer lipopolysaccharide membranes through P & L rings respectively. Gram positive bacteria lacks outermost ring (P & L). Flagellum consists of protein called FLAGELLIN.

47. Flagellar types of bacteria

48. 2. PILI (FIMBRIAE) They are hollow, non-helical, filamentous appendages that are thinner, shorter & more numerous than flagella. It occurs in both flagellated & non flagellated bacteria. Each bacteria possess 100-200 peritrichously borne pili. They are 1-1.5 micrometers in length & 4-8 nm in diameter. They are made up of protein PILIN.

49. FUNCTIONS F-pilus (fertility pilus) or sex pilus which involved in bacterial conjugation helps in transfer of genetic material between bacterial cells. Some pili allow the pathogenic bacteria to attach to epithelial cells lining the respiratory, intestinal or genitourinary tract & thus establish an infection. Eg:- Neisseria gonorrhoeae

50. 3. CAPSULE / SLIME LAYER The high molecular substances produced by bacteria during their active growth gets collected on surface of cells & forms a gelatinous covering around these cells & when these covering does not form a persistent layer but is present more diffusely forming a loose mass or matrix around the bacterial cell, it is called SLIME LAYER or BIOFILM. When this gelatinous layer forms a well developed persistent layer, it is called CAPSULE.

51. Bacterial capsules are composed of either polysaccharide (Eg:- Klebsiella pneumonia) or polypeptide (Eg:- Bacillus anthracis) FUNCTIONS Provides protection against temporary drying by binding water molecules. It may block attachment of bacteriophages

52. It may inhibit engulfment of pathogenic bacteria by phagocytes & thugs contribute to virulence of bacteria. It may promote attachment of bacteria to smooth surfaces . Eg:- Streptococcus mutans. It may protect bacteria from antibacterial agents such as lytic enzymes found in nature. Bacteria over produces capsular material when overfed with sugars, become reserves of carbohydrates for subsequent metabolism.

53. 4. CELL WALL It is the rigid structure surrounding the plasma membrane. Present in all prokaryotes except Mycoplasma & Methanoplasma. Peptidoglycan (Murein, Muramic acid, Mucopeptide) is the main constituent of bacterial cell wall. Peptidoglycan It is an insoluble , porous, cross linked polymer of enormous strength & rigidity. It is found only in prokaryotes & founds as a “bag shaped macromolecule” surrounding the cytoplasmic membrane.

54. Peptidoglycan consists of 2 parts:- A peptide portion : composed of 4 amino acids (L-alanine, D-glutamine, either L-lysine or diaminopimelic acid & D-alanine). Glycan or sugar portion : Made up of two alternating units of N-acetyl glucosamine (NAG) & N-acetyl muramic acid (NAM). Peptidoglycan is present in gram positive & negative bacteria.

55. Cell wall of gram positive bacteria Cell wall appears as a thick homogeneous layer. Consists of peptidoglycan. Remaining is made up of proteins, polysaccharides & teichoic acid. Teichoic acid affect the passage of ions thus maintaining low pH of cell wall so that autolysins don't degrade the cell wall.

56. Cell wall of gram negative bacteria Cell wall is more complex than gram positive. Contains an outer membrane that surrounds a thin underlying layer of peptidoglycan. Rich in lipids. Outer membrane is attached to peptidoglycan by means of “ Braun’s lipoprotein”. This membrane is bilayered mainly of phospholipids, proteins & lipopolysaccharides (LPS). LPS has toxic properties & known as Endotoxins which is released only after lysis of cell wall.

57. Outer membrane serves as impermeable barrier to prevent escape of important enzymes. It also prevents the entry of various external chemical & enzymes that could damage the cell wall. Its impermeable to larger molecules (protein) but permeable to smaller molecules (amino acids, peptides) to pass across by means of channels in special proteins called “porins”.

58. Functions of cell wall The cell wall being rigid structure, gives shape to the cell. Cell wall prevents the cell from expanding & bursting, when osmotic pressures are exerted on it.

59. Structures internal to cell wall CYTOPLASMIC MEMBRANE / PLASMA MEMBRANE Present just beneath the cell wall Thickness : 7.5 nm Composed of phospholipids & proteins. Ultra structure of cell membrane has been explained by Fluid Mosaic Model proposed by Singer & Nicolson in 1974. The phospholipids form a bilayer in which most of the proteins are firmly held called INTEGRAL or INTRINSIC PROTEINS which can be removed only by destruction of membrane.

60. Other membranes called PERIPHERAL or EXTRINSIC PROTEINS are loosely attached & can be removed by mild treatments such as osmotic shock.

61. Functions of plasma membrane It act as a differentially permeable barrier, regulating the flow of materials in & out of the cell. Transport proteins in the membrane facilitates the passage of small molecules ( nutrients & wastes) across the membrane . It contains various enzymes involved in respiratory metabolism & in synthesis of cell wall, septum formation , membrane synthesis & DNA replication. It is the site of proton motive force. It contains specific attachment sites for chromosomes & for plasmid that plays vital role at the time of cell division.

62. Difference between flagella & fimbriae. Difference between gram positive & negative bacterial cell wall.

63. 2. CYTOPLASM It is a homogeneous aqueous solution bounded by cell membrane & is divided into 3 distinct areas. Cytoplasmic area :- Granular in appearance & rich in ribosomes in which proteins are synthesized. Chromatin area :- Rich in DNA Fluid portion :- Consisting of dissolved substance ( cell solutes, metabolites) It lacks endoplasmic reticulum, golgi apparatus, mitochondria & a true bound nucleus.

64. 3. INCLUSIONS Present in prokaryotic cells Theory are distinct granules that may occupy a substantial part of the cytoplasm.

65. 4. MESOSOMES They are characteristic infoldings in the form of system of convoluted tubules & vesicles present especially in gram + bacteria. On the basis of their location they may be:- Central - Central mesosomes penetrate deeply into the cytoplasm & are located near the middle of cell & appear to be attached with nuclear membrane. Involved in DNA replication & septa formation. Peripheral - They do not penetrate into cytoplasm & are located near periphery. They are involved in export of exocellular enzymes like penicillinase.

66. 5. NUCLEAR MATERIAL Does not contain membrane bound nucleus or mitotic apparatus. Known as Nucleoid, the chromatin body, the nuclear equivalent & bacterial chromosome. Its a large circular molecule of DNA , more or less free in cytoplasm although coiled & super coiled anchored by proteins. The total DNA content of a prokaryote is referred to as Cell Genome. The DNA can be visible under light microscope by Feulgen staining which is specific to DNA.

67. 6. ENDOSPORES Spores are highly resistant, metabolically dormant structures produced by certain bacteria during their unfavourable environmental conditions. Since these spores are formed within the parent cell, they are called Endospores. Endospores are thick walled, highly refractile bodies that are usually produced by cells growing rich in culture media when approaching the end of active growth. Eg:- Bacillus ( B. anthracis, B. subtilis), Clostridium (Cl. tetani , Cl. botulinum), Sporosarcina, Thermoactinomyces

68. Morphology of spore

69. Formation of spore (Sporulation)

70. Shape & Position of Spore

71. Resistance of spore :- endospores are resistant to ordinary boiling, heating & disinfectants. They can withstand boiling upto 3 hrs, dry heat at 150 deg for 1 hr. They can be destroyed by autoclaving at 121 deg C for 15-20 minutes. Germination of spore :- The conversion of spore into vegetative cell under favourable environmental conditions is called germination & it may occur less than 2 hrs.

72. Germination of bacterial spore 3 stages:- ACTIVATION :- Activated by heat (60 deg C for 1 hr), low pH (acidic), abrasion that can damage the spore coat. INITIATION :-Binding of effector substances to spore coat, which activates autolysins that destroy peptidoglycan of cortex, allowing uptake of water & release of calcium dipicolinate. OUTGROWTH :- With disintegration of cortex & swelling of spore , a single germ cell emerges after breaking open the spore coat. The new vegetative cell contains spore protoplasts which is followed by a period of biosynthesis producing an outgrowth that transfers to new vegetative cell.

73. C) Growth & Nutrition of Microbes

74. Bacterial growth involves:- Increase in cell mass Duplication of DNA Synthesis of new cell wall & cellular membrane Cell division by BINARY FISSION

75. Nutritional types of bacteria On the basis of Carbon & Energy sources for growth, bacteria are classified into 2 groups :- Autotrophic bacteria Photoautotrophic bacteria Chemoautotrophs or Chemosynthetic bacteria Heterotrophic bacteria Photoheterotrophs Chemoheterotrophs

76. Physical / Environmental Factors Affecting Growth Of Bacteria TEMPERATURE Psychrophiles / Cryophiles Obligate / Strict Psychrophiles :- can't grow above 20-22 deg C of temperature & dies at room temperature. Facultative Psychrophiles :- grow at 0 deg C, but growth is best at a range of 20-30 deg C.

77. Mesophiles Thermotolerants / Thermoduric :- they can withstand with high temperature but can’t multiply. Psychro / Cryotolerants :- also called psychroduric or cryoduric. They can survive at very low temperature but don’t grow & multiply. Thermophiles Obligate thermophiles :- they grow only at temperature above 50 deg C, & can’t grow in mesophilic range. Facultative thermophiles :- they can grow both at high temperature & in mesophilic range. (37-50 deg C).

78. 2. OXYGEN Aerobes Anaerobes Obligate aerobes Obligate anaerobes Facultative aerobes Facultative anaerobes Capnophilic :- these are organisms which can grow best in an atmosphere of 5-10 % of carbon dioxide . Microaerophilic :- they require low levels of oxygen but can’t tolerate the level of oxygen in the atmosphere.

79. 3. HYDROGEN ION CONCENTRATION (pH) Acidophiles :- microbes which grow at an optimum pH well below neutrality (7.0). Neutrophiles :- Microbes which grow best at neutral pH. Alkaliphiles :- Microbes that grow best under alkaline conditions.

80. 4. OSMOTIC PRESSURE Osmophobic Osmophilic Halophilic Osmoduric 5. LIGHT Bacteria except phototrophs grow in darkness.

81. 6. WATER Water activity of pure water is 1.0 Bacteria live over a range of water activity from 1.0 - 0.7 7. CARBON DIOXIDE

82. BACTERIAL GROWTH CURVE A characteristic growth curve obtained in batch culture ( culture in which no fresh nutrients are added) consists of four phases. LAG PHASE LOG / EXPONENTIAL PHASE STATIONARY PHASE DEATH / DECLINE PHASE

83. GROWTH CURVE OF BACTERIA IN BATCH CULTURE

84. LAG PHASE Bacteria adapts themselves to growth conditions. Phase of intense metabolic activity (prepare for reproduction, DNA synthesis, cell division) Increase in size (volume) of bacterial cell but no increase in cell number. Length of this bacteria depends upon the bacteria & quality of cell culture media.

85. 2. LOG / EXPONENTIAL PHASE Bacteria multiply in its maximum rate & their number increases exponentially or by geometric progression. Plotting the natural logarithm of cell number against time produces a straight line. Duration of this phase is limited since the bacteria is growing in a constant medium of batch culture.

86. 3. STATIONARY PHASE Growth rate slows down as a result of nutrient depletion & accumulation of inhibitory end (toxic) products. Growth rate becomes equal to death rate rate.

87. 4. DEATH / DECLINE PHASE Death rate exceeds reproduction rate & thus no. of viable bacterial cells start declining. Entire bacterial population dies after viable period & culture becomes sterile.

88. D) BLOOD & BODY FLUIDS

89. Composition of Blood The two main constituents of blood are liquid plasma or blood serum & the various types of cells which floats in it. Composition of blood serum Water : Main constituent. 90% of blood is water. Dissolved gases : Oxygen , CO2, Nitrogen Dissolved solids : plasma proteins like fibrinogen, globulins, albumins, antibodies, enzymes, hormones & waste materials. Inorganic substances like Fe, Ca, Mg, Na, K, Chlorides , phosphates , carbonates, bicarbonates, sulphates.

90. Nutrients : fatty acids, glucose, amino acids, cholesterol & fats. Excretory materials : Urea, Ammonia, Uric acid & Creatinine Blood corpuscles (formed elements) : RBC, WBC, Thrombocytes or Platelets.

91. Functions of Blood Transport of nutrients Transport of excretory products Transport of respiratory gases Transport of hormones Protection against infection Clotting of blood Maintenance of temperature Regulation of pH & water balance Maintenance of physiological state of body Transport of organic & inorganic substances

92. Disorders of Blood Anemia Hemophilia Polycythemia Leukemia

93. Body Fluids They are dilute watery solutions containing dissolved chemicals that are found inside cells (Intracellular fluid or ICF) as well as surrounding them (Extracellular fluid or ECF). The ECF that fills the narrow spaces between cells of tissues is called Interstitial fluid. ECF within blood vessels - Plasma ECF within lymphatic vessels - Lymph

94. ECF in & around brain & spinal cord - Cerebrospinal fluid(CSF) ECF within joints - Synovial fluid ECF within eyes - Aqueous humour & Vitreous body The proper functioning of the body cells depends on well regulation of composition of interstitial fluid often called internal environment. Serous fluids - Pleural, Pericardial & Peritoneal fluids.

95. E) Laboratory Methods For Identification Of Microbes

96. Steps for isolation & identification of pathogenic bacteria Collection of specimen Direct microscope examination Dry mount lamination (preparation of smear, fixing of smear & staining) Culture Observation of microbes Motility test Biochemical reactions Serological diagnosis Animal pathogenicity Antibiotic sensitivity test Phage typing

97. Specimen Collection Precautions in specimen collection Specimen should be collected under aseptic technique. Specimen should contain only those organisms from the site where it was collected. Avoid contaminating discharge or ulcer material with skin commensals . Specimen should be collected in dry sterile, leak proof container free from all traces of disinfectants.

98. Every specimen should accompanied by request for which include the information like patient’s name, date & time of collection, ward details if it is collected in hospital , investigation required, clinical notes. High risks specimens like HIV, Hepatitis B must be handled with extra care & should be mentioned as HIGH RISK SPECIMEN.

99. Sl.No: Specimen Container Patient preparation Transport to lab Storage 1 Urine (midstream) Sterile screw cap with wide mouthed bottle Male : clean glans & collect midstream urine Female : clean area around urethral opening & collect midstream urine Within 2 hrs at 2-8 deg C 24 hrs at 2-8 deg C 2 Stool Clean or leak proof screw cap with wide mouthed bottle No specific preparation Within 24 hrs at 2-8 deg C 72 hrs at 2-8 deg C 3 Rectal swab Swab placed in enteric transport medium Insert 2.5 cm past anal sphincter Sterile screw cap with wide mouthed bottle 72 hrs at 2-8 deg C

100. 4 Blood & bone marrow Vacutainer tube with polyanethol sulphate Draw blood during sterile episode & collect two sets from left & right arm. Within 2 hrs at room temperature Should be incubated at 37 deg C 5 Sputum Sterile screw cap with wide mouthed bottle Collected after deep coughing Within 2 hrs at room temperature 24 hrs at room temperature 6 CSF Sterile screw cap tube or bottle Disinfect skin before aspirating Keep immediately at temperature 6 hours at 37 deg C 7 Body fluids Sterile screw or anaerobic transport Disinfect skin before aspirating Keep immediately at room temperature Prepare plate as soon as received 8 Skin scrapings Anaerobic transporter Disinfect skin, do not allow to dry Within 24 hrs at room temperature 24 hrs at room temperature

101. Direct microscopic examination First step in laboratory diagnosis of pathogenic organism. It is useful when organism cannot be cultured i the lab.this may be done with a wet unstained specimen or with a dry fixed stained specimen. Two methods are :- Wet mount method Hanging drop method

102. WET MOUNT METHOD It is done without fixing the specimen. It helps in the identification of motile organisms, fragile structures & sporulating bodies. Stained smears are also used to study staining characters. Specimens that are used are:- urine sediment, fecal suspension, skin scrapings. METHOD Transfer a loopful of the specimen to a microscopic slide. Put a drop of saline & place the cover slip. Avoid air bubbles. Examine slide under microscope with low power objective (100 X magnification).

103. 2. HANGING DROP METHOD It is a method in which a drop of bacterial suspension is enclosed in an airtight chamber prepared in a special depression / concavity slide. It is used to examine the motility of bacteria in a given culture . Most frequently used to examine the stool specimen of suspected Cholera patients.

104. Observe the slide through the eyepiece . Focus the edge of the drop carefully. The cells will look either dark or slightly green, very small rods or spheres . Observation :- BROWNIAN movement should be visible on slide indicating motility of given organism.

105. STAINING TECHNIQUES Stains are dyes or reagents used for differential colouring of microbes to observe their structures with much clarity under microscope. They may be either:- ACIDIC DYES BASIC DYES NEUTRAL STAINS DRY MOUNT LAMINATION :- It is the examination of the stained smear. Morphological features are studied better through this method.

106. Purpose of staining To view the organism with much clarity. To determine peculiar structures. To differentiate one organism from another.

107. Types of Stains DIRECT / GENERAL STAIN INDIRECT STAIN SELECTIVE STAINS DIFFERENTIAL STAINS They are able to stain bacteria with exceptions of bacterial spore & bacteria with waxy coating on their cell wall. They stain only the background. Eg:- Nigrosin or India ink They are used to stain particular part of the organism like spores, flagella , nuclei etc. They differentiate two groups of bacteria in a mixture. (gram positive & negative )

108. Staining Techniques / Procedures There are two staining procedures for light microscopy:- SIMPLE STAINING DIFFERENTIAL STAINING SIMPLE STAINING Single staining reagent is used ( methylene blue, crystal violets , carbol fuchsin). All cells & structures stain in the same manner. Its of two types. (Positive staining & Negative staining)

109. i) Positive staining :- Here the stain ( methylene blue) is basic (cationic) & attaches to the surface of the object that are negatively charged. ii) Negative staining :- The stain(India ink, Nigrosin) is acidic (anionic). Bacterial cell appears transparent & only background is stained.

110. Differential staining More than one staining reagents are used. Specific objects exhibit different staining reactions which are distinguishable. Main two differential staining procedures are Gram staging & Acid fast staining.

111. Simple Staining Of Bacteria Principles Bacterial smear is stained with single dye or staining reagent. Purpose is to demonstrate cell size, shape & arrangement of bacterial cells. Bacterial cell surface is negatively charged, so it tends to bind strongly to the cationic chromogen of basic dyes. Reagent :- Loeffler’s methylene blue solution

112. Procedure ;- Prepare a smear of a given culture by spreading a thin film on a clean glass slide. Dry it by waving in air & then heat fix by passing the slide 2-3 times through the flame wit the smeared side facing upwards. Stain the smear by flooding it with one of the staining solutions & allowing it to remain covered with the stain for the time designated below;- METHYLENE BLUE (blue) - 1 minute CRYSTAL VIOLET (purple) - 30 seconds CARBOL FUCHSIN (red) - 20 seconds

113. Wash the slide gently with running water to remove excess stain. Air dry the s lides or blot dry with blotting paper. Apply oil directly to the smear & focus the smear under low power objective & then under oil immersion objective. Results The bacteria will appear blue or red depending upon the stain used with characteristic morphology .

114. Gram Staining Of Bcateria The technique was developed by Danish physician, Dr. Hanes Christian Gram (1884). Gram staining determines gross morphology, differentiate bacteria into two major distinct groups, ie; Gram Positive & Negative . It involves 6 basic steps;- Smear preparation, heat fixing of smear, staining with crystal violet (primary stain), use of Iodine/Lugol’s iodine (mordant), treatment with acetone alcohol mixture (decolourizing agent) & use of Safranin (counterstain).

115. Principles In gram negative bacteria the cell wall is thin, multilayered containing high lipids which are readily dissolved by alcohol, resulting in pore formation of the cell wall facilitating the leakage of the crystal violet-iodine complex & resulting in discoloration of gram negative bacteria & takes up safranin & appears red.

116. Cell walls of grampostive bacteria is thick, composed mainly of proteins & cross linked mucopeptides. On the application of decolourizing agent, dehydration is caused resulting in closure of pores of cell wall thereby retaining the primary stain colour & appears as blue or purple. Reagents Crystal violet stain Gram iodine solution Ethyl alcohol (95%) or alcohol-acetone (1:1) solution Safranin solution

117. Procedure Make smear of a given culture on a clear glass slide . Air dry the smear & heat fix it. Cover the smear completely with crystal violet & leave the stain on the slide for 1 minute. Wash the slide gently in distilled water or tap water. Flood the smear with Gram or Lugol’s iodine solution & wait for 1 minute. Wash with tap water gently & drain carefully. Add ethyl alcohol or alcohol- acetone solution drop by drop, until the smears becomes free from any colorization.

118. Wash the slide gently under tap water & drain. Now counterstain with safranin & wait for 30 seconds . Wash again & blot dry with blotting paper or simply air dry & observe under oil immersion objective . Results Bacteria that appear BLUE /VIOLET/PURPLE are assigned as Gram Positive & those appearing as RED/PINK as Gram Negative.

119. ACID FAST STAINING OF BACTERIA Technique was developed by Paul Ehrlich (1882) & was modified by Ziehl-Neelsen. Hence known as Ziehl - Neelsen stain. It's a differential stain & is used to identify mainly the Mycobacterium especially Mycobacterium tuberculosis & Mycobacterium leprae. Bacteria are classified into :- Acid fast & Non acid fast organisms.

120. Reagents used:- Carbol fuchsin solution (Primary stain) Acid-alcohol solution (3% HCl in alcohol) Methylene blue (counter stain) Results:- Acid fast organisms will appease bright red on a blue background while non acid fast organism will appear dark blue in colour.

121. CULTURE MEDIA It is a solid or liquid preparation for cultivating microbes by letting them to reproduce in a predetermined laboratory conditions (giving adequate nutrients & meeting all growth requirements).

122. Classification of Culture Media Classification based on nature of ingredients The media which require living cells or tissues which are parasitized by microbes to be cultured. Eg: Rickettsiae The media that doesn't require living cells or tissues . This media can be divided into two:- i) Synthetic media /Defined media ii) Non-synthetic media /Complex media

123. Synthetic media /Defined media These are the media in which only pure chemicals in definite concentrations are used. These are useful for nutritional & metabolic studies. Non- synthetic media / Complex media These are the media in which exact chemical compositions of each of the constituent is not known with certainty .

124. B) Classification based on consistency (physical state) of medium Liquid M edia :- These are used in liquid form. Eg:- Nutrient broth, Brucella broth, Nitrate broth. Semisolid Media :- These media contain 0.5% or less of agar imparting custard consistency to the medium. Eg:- Cysteine Trypticase agar medium.

125. 3. Liquefiable Solid Media :- This media is prepared by adding suitable amount of gelatin or agar to the liquid medium to remain solid when cool but become liquid when warm or vice versa. Hence its also called Solid reversible to Liquid media. Eg:- Salmonella - Shigella Agar, Nutrient Gelatin Medium, Blue-Esculin Agar Medium. 4. Solid Media :- These media always remain olid. Eg:- Nutrient Agar Medium, Bile Salt Agar Medium.

126. C) Classification based on application or function Cultivation media Storage media Enrichment media Differential media Selective & selective differential media Assay media 7. Maintenance media 8. Transport media 9. Enumeration Media 10. Anaerobic media 11. Sugar media

127. CULTIVATION MEDIA :- Used for general cultivation of bacteria. Eg:- Nutrient Broth/Agar STORAGE MEDIA :- Bacterial cultures are preserved & stored for longer periods of time by Lyophilization (deep - dry method). ENRICHMENT MEDIA :- These are the media in which nutritional environment is adjusted in such a way that the growth of certain bacterial types is enhanced selectively within a mixed population. Eg:- GN Broth, Salenite F, Loefller’s serum slope.

128. 4. DIFFERENTIAL / INDICATOR MEDIA :- These are the media which differentiate two organisms by their characteristic reactions towards the medium like haemolysis or biochemical changes. Eg:- Blood -Agar medium which differentiates haemolytic species from non-haemolytic species, MacConkey Agar or EMB Agar differentiates lactose fermenters from non lactose fermenters.

129. 5. SELECTIVE & SELECTIVE DIFFERENTIAL MEDIA i) Selective media :- this media allows the growth of selective groups of organisms while inhibiting others by various inhibitors like antibiotics, dyes, bile salts. Eg: MacConkey Agar for isolation of E.coli, Deoxycholate citrate agar for Salmonella & Shigella, Lowenstein-Jensen medium for M.tuberculosis.

130. ii) Selective differential media :- This media allows the selective growth of certain organisms & also differentiation of various groups of organisms on the basis of their biochemical reactions in a particular media. Eg:- Blood Agar for isolation of bacterial pathogen (S. pyogenes & S.pneumoniae)

131. 6. ASSAY MEDIA :- Also called Media for Special Purpose. This has got profound influence on the bacterial cells with respect to formation of enzymes, toxins, antibiotics & other bioactive products. Its used to test the effectiveness of antimicrobial drugs & to assess the effect of disinfectants, antiseptics, cosmetics & preservatives on the growth of microbes. Eg:- Pyridoxine deficient growth medium for Streptococcus faecalis . 7. MAINTENANCE MEDIA :- its used to maintain physiological characteristics & hence viability of bacterial cultures.

132. 8. ENUMERATION MEDIA :- They are used primarily to count the number of microbes in milk, water, food & other samples & find their broad application in concerned industries. 9. ANAEROBIC MEDIA :- These media are used for the growth of anaerobes. Eg:- Robertson’s cooked meat medium or Thioglycolate broth for isolation of Clostridium species. 10. SUGAR MEDIA :- This media carries out sugar fermentation, which is important for the identification of most of the microbes.

133. 11 . TRANSPORT MEDIA :- These media inhibit self destruction & multiplication of microbes & hence maintains the specimen in a “status quo”. Used for Neisseria species. Different transport media for transporting different specimens Cary-Blair & Amies transport media :- used for transporting fecal material on rectal swab & are effective in preserving Salmonella, Shigella & Vibrio. Stuart transport media :- used for CSF & cervical specimens. Preserves both Neisseria species & Trichomonas vaginalis for 18-24 hrs.

134. c) Venkataraman - Ramakrishnan (V.R) media :- used to transport stool specimen of suspected cholera patient. d) Bile Peptone transport medium :- used to transport stool specimen of suspected cholera patient. Useful medium for field works when more delay is expected. e) Brucella Broth :- Used to transport biopsy specimen from duodenal ulcer patients. The medium preserves the etiological agent under refrigeration for 24-48 hrs.