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1. INTRODUCTION Antony Van Leeuwen hoek (1632-1723) Spontaneous germ theory Francesco Redi (1626 – 1697) John Needham (1713 -1781) Lazzaro Spallanzani (1729-1799) Theodore Schwaan (1810-1882) Georg Friedrich Schroder and Theodor Van Dusch Louis Posteur (1822 – 1895)

2. Koch postulate’s The micro organism must be present in every case of the disease but absent from healthy organism. The suspected micro organism must be isolated and grown in a pure culture. The same disease must result when the isolated microorganism is inoculates from the healthy host. The same microorganism must be isolated again from the diseased host.

3. Scope and relevance of microbiology Medical microbiology Public health microbiology Immunology Agricultural microbiologist Microbial ecology Food and dairy microbiology Industrial microbiology Microbial physiology Microbial genetics and molecular biology

4. Microbes Classification means the orderly arrangement of units under study into groups of larger units. In 1866 E.H.Haeckel suggested a third kingdom that includes those unicellular microorganisms that are typically neither plant nor animals. These organisms, the protists, include bacteria, algae, fungi and protozoa. Bacteria are referred to as lower protists; the others – algae, fungi and protozoa are called higher protists. In typical bacteria, the nucleus was not enclosed by a nuclear membrane. In other cells, such as typical algae and fungi, the nucleus was enclosed in a membrane. These two cell types have been designated prokaryotic and eukaryotic Bacteria are prokaryotic micro organisms. The eukaryotic microorganisms include the protozoa, fungi, algae, plants and animals.

5. Differences Between Eucaryotic and Procaryotic Cells Procaryotes Eucaryotes Cell size 0.2-2 um in diameter 10-100 um in diameter Nucleus Absent Present Membranous Organelles Absent Present Cell Wall Chemically complex When present, simple Ribosomes Smaller (70S) Larger (80S) in cell 70S in organelles DNA Single circular Multiple linear chromosome chromosomes (histones) Cell Division Binary fission Mitosis

6. MODE OF NUTRITION PHOTOSYNTHETIC INGESTIVE ABSORPTIVE

7. Five-Kingdom Classification System

8. Five-Kingdom System of Biological Classification Proposed in 1969 by Robert Whitaker : 1. Kingdom Procaryotae ( Monera ): Oldest known cells. Lived over 3.5 billion years ago. Lack a nucleus and membrane bound organelles. The other four kingdoms are eucaryotes . Have a true nucleus and membrane bound organelles. 2. Kingdom Protista : Mostly unicellular, lack tissue organization. Most have flagella during life. 3. Kingdom Fungi : May be unicellular (yeasts) or multicellular (molds). Many are saprotrophs . 4. Kingdom Plantae : Multicellular , photosynthetic. 5. Kingdom Animalia : Multicellular , heterotrophs that ingest food through a mouth or oral cavity.

9. Classification of Organisms Hierarchy of Taxonomic Categories DOMAIN Kingdom Phylum or Division (Bacteria) Class Order Family Genus species

10. Taxonomy Taxonomic group of higher rank than genus are Family A group of similar genera Order A group of similar families Class A group of similar orders Division A group of similar classes Kingdom A group of similar divisions

11. Bacteriology – Study of bacteria Protozoology – Study of protozoa Mycology -- Study of fungi Virology - It is the science which deals with viruses Phycology is the study of algae

12. The prokaryotes are included in the kingdom Monera : they lack the ingestive mode of nutrition. Unicellular eukaryotic microorganisms are placed in the kingdom protista ; all three nutritional types are represented here. The multicellular and multinucleate eukaryotic organisms are found in the kingdoms Plantae . ( multicellular green plants and higher algae) Animalia ( multicellular animals). Fungi (multinucleate higher fungi). Microorganisms are found in three of the five kingdoms: Monera (bacteria and cyano bacteria), Protista (micro algae and protozoa), and Fungi (yeasts and molds).

13. Groups of micro organisms The major groups of protists are Bacteria: Unicellular prokaryotic organisms multiplied by binary fission. Protozoa: Unicellular eukaryotic organisms and are differentiated on the basis of morphological, nutritional and physiological characteristics. Fungi: Eukaryotic lower plants devoid of chlorophyll. Usually multicellular but are not differentiated into roots, stem, and leaves. They range in size and shape from single celled microscopic yeast to giant multicellular mushrooms and puff balls.

14. Algae: Unicellular, contain chlorophyll and are capable of photosynthesis. Rickettsiae : obligate intracellular parasites rod shaped coccoid or pleomprphic with typical gram negative walls with no flagella. 0.3 to 0.5 micrometer in diameter and 0.8 to 2.0 micro meter long. Spirochetes : spiral shaped prokaryotes can be either classified as spirilla have more flexible and internal flagellar arrangement.

15. True fungi are composed filaments and masses of cells which make up the body of the organism, known as mycelium. Fungi reproduce by fission, by budding, or by means of spores borne on fruiting structures that are quite distinctive for certain species. Viruses: They are included for two reasons 1) the techniques used to study viruses are microbiological in nature 2) viruses are causative agents of diseases. They are very small noncellular parasites or pathogens of plants, animals, and bacteria as well as other protists. They can be visualized only by electron microscope. Virus can be cultivated only in living cells.

16. Bacteria Prokaryotes represent two domains, bacteria and archaea . Archaea live in Earth’s extreme environments. Bacteria are the most abundant and diversified organisms on Earth. Bacteria is an singular or unicellular prokaryotic organism.

17. Classification of microorganism The micro organisms classified in different categories from different view points. On the basis of their body and nuclear organization On the basis of organisms classification On the basis of nutrition On the basis of Oxygen requirement On the basis of temperature requirements On the basis of distribution On the basis of economic importance On the basis of osmotic conditions

18. On the basis of their body and nuclear organization basis of their body organization. i ) Microbes beyond cellular organization ii) Cellular micro organisms. basis of nuclear organization the cellular microbes can further be grouped in to two ( i ) Prokaryotes ( Archaebacteria , cyanobacteria and eubacteria ). (ii) Eukaryotes ( algae, protozoan, slime molds and micro fungi). On the basis of organisms classification Whittaker’s five kingdom classification On the basis of nutrition ( i ) Photo auto trophs - use light energy to manufacture their food. (ii) Chemo auto trophs - use chemical energy to manufacture their food. (iii) Photoheterotrophs - utilize the light energy in obtainment (absorption) of their food from external environment; (iv) Chemo heterotrophs - use chemical energy for in obtainment of their food from external environment;

19. On the basis of Oxygen requirement Aerobes Obligate aerobes Facultative aerobes Anaerobes Obligate anaerobes Facultative anaerobes On the basis of temperature requirements Psychrophilic or Cryophilic - 0°C temperature as the  minimum, 15 – 20 ° as optimum and 30° as maximum. Mesophile -minimum, optimum and maximum temperature limits are 15-25°, 25- 40° and 50° respectively. Thermophile -minimum temperature limit is 25-45°C; optimum as 45-55° C and maximum limit is 55- 85°C. Thermo tolerants or thermoduric Psychro or cryo tolerants

20. On the basis of distribution Hydrosphereic or aquatics Lithospheric or terrestrial Atmospheric or aerial On the basis of economic importance i ) Useful microbes – Extend benefits to us. (ii) Harmful microbes – Cause harm or losses directly. On the basis of osmotic conditions ( i ) Osmophobic : (ii) Osmophoric (iii) Halophilic (iv) Osmoduric

21. Bacteria Unicellular 0.5 to 5 µm in length. A few species–for example Thiomargarita namibiences and Epulopiscium fishelsoni –are up to half a millimetre long and are visible to the unaided eye. Mycoplasma - 0.3 micrometers. Bacterial species are either spherical, called cocci or rod-shaped, called bacilli.

22. Classification of Bacteria Scientific Nomenclature Bacterial species : Population of cells with similar characteristics. Bacterial strain : A subgroup of a bacterial species that has distinguishing characteristics. Identified by numbers, letters, or names that follow the scientific name. Escherichia coli O157:H7 : Strain that causes bloody diarrhea. Bergey’s Manual: Provides a reference for identifying and classifying bacteria. Classification initially based on cell morphology, staining, metabolism, biochemistry, serology, etc. More recently, DNA, RNA, and protein sequence analysis are being used to study evolutionary relationships.

23. Bacteria Have One of Three Cellular Shapes Rods (bacilli) Coccoid -Shaped Spirilla

24. Arrange ment

25. Structure of bacteria

26. Bacterial Morphology   Cell envelope: In gram positive plasma membrane, the cell wall and in gram negative an outer membrane that is the part of the cell wall. Cell envelope consists of two components, a rigid cell wall and beneath it a cytoplasmic or plasma membrane. Protoplasm : cytoplasm, cytoplasmic inclusions such as ribosomes and mesosomes, granules and vacuoles and the nuclear body. Structure outside the cell wall capsule, slime layers and S- layer. Flagella which are organs of locomotion Fimbriae which appear to be organs of adhesion.  

27. Capsule and S-layer Some of the bacterial cells are surrounded by the extra cellular polymeric substances called capsule. A slime layer is a zone of diffuse, unorganized material that is removed easily. A glycocalyx is a net work of polysaccharides extending from the surface of bacteria and other cell. The capsule is a gelatinous polymer made up of either polysaccharides ( Klebsiella pneumoniae ) or poly peptide (Bacillus anthracis consists of poly-D-glutamic acid). Capsules are clearly visible in the light microscope when negative stain or special capsule stains are involved. Streptococcus pneumoniae provides a classic example, when it lacks a capsule it is easily destroyed by enzymes and does not cause disease, where as the variant quickly kills mice. The glycocalyx also aids bacterial attachment to the surfaces of solid objects in aquatic environments or to tissue surfaces or in animals.

28. Functions of capsule The capsule may prevent the attachment of bacteriophages. It protects the bacterial cell against the desiccation as it is hygroscopic and contains water molecules. It may survive in natural environment due to its sticky property. After attachment they can grow on the diverse surfaces. They may prevent the engulfment by WBCs and therefore contribute to virulence S.mutans uses it capsules as sources of energy. It breaks down the sugars of capsule when stored energy in low amount. Capsule protects the cell from desiccation, maintains the viscosity and inhibits the movement of nutrients from the bacterial cell.

29. Structure of cell wall Rigid and one of the most important parts of a prokaryotic cell for several reasons. Two kind of bacteria based on Gram reaction -Gram positive another one is Gram negative. The gram positive cell wall consists of a single 20 to 80nm thick homogenous peptidoglycan or murein layer lying outside the plasma membrane. In contrast, the gram negative cell wall has a 2 to 7 nm peptidoglycan layer surrounded by a 7 to 8 nm thick outer membrane. Because of the thick peptidoglycan layer the walls of gram positive cells are stronger than those of gram negative bacteria. A space is seen between plasma membrane and the outer membrane, this space is called the periplasmic space.

30. Structure of peptidoglycan Peptidoglycan or murein is a polymer contain two sugar derivatives, N- acetyl glucosamine and N-acetyl muramic acid , amino acid such as -- D- glutamic acid , D- alanine and meso diamino pimelic acid . The presence of D- amino acids protects against attack by most peptidases. A peptide chain of four alternating D- and L- amino acids connected to the carboxyl group of N-acetyl muramic acid. Many bacteria substitute another diamino acid L-lysine, in the third position of meso - diamino pimelic acid. Often the carboxyl group of the terminal D-alanine is connected directly to the amino group of diamino pimelic acid, but a peptide interbridge ( pentaglycine bridge)may be used instead. Most gram negative cell wall peptidoglycan lacks the interbridge .

31. Gram positive cell wall

32. Gram positive cell wall Gram positive cell wall contain large amount of teichoic acids, polymer of glycerol or ribitol joined by phosphate group. Amino acids such as D- alanine or sugars like glucose are attached to the glycerol or ribitol group. The teichoic acids are connected to either the peptidoglycan itself by a covalent bond with the six hydroxyl of N- acetyl muramic acid or to plasma membrane lipid; in the later case they are lipoteichoic acid. Staphylococci and most other gram- positive bacteria have a layer of proteins on the surface of their cell wall peptidoglycan . In Staphylococci, these surface proteins are covalently joined to the Pentaglycine Bridge of the cell wall peptidoglycan .

33. An enzyme called sortase catalyzes the attachment of these surface proteins to the gram positive peptidoglycan . Sortase is attached to the plasma membrane of the bacterial cell. Peptidoglycan can be extracted with hot dilute hydrochloric acid and teichoic acid extracted with cold dilute acid. The walls of Mycobacterium, Corynebacterium , being rich in lipid called Mycolic acid. The ability of mycobacteria to exhibit acid fast staining is correlated with the presence of mycolic acids. A mycolic acid derivatives called cord factor ( trehalose dimycolate is toxic and plays an important role in the disease caused by C.diphtheriae and M. tuberculosis.

34. Gram negative cell wall

35. Gram negative cell wall

36. The thin peptidoglycan layers may constitute not more than 5 to 10%of the wall weight. The outer membrane lies outside the thin peptidoglycan layer. Braun’s lipoprotein, a small lipoprotein covalently attached to the underlying peptidoglycan and embedded in the outer membrane by its hydrophobic end. The outer membrane serves as a barrier to various external chemicals and enzyme that could damage the cell. For example the wall of the many gram positive bacteria easily destroyed by treatment with an enzyme called lysozyme, which selectively dissolves peptidoglycan. Another structure which strengthens the gram negative wall and holds the outer membrane in place is the adhesion site.

37. The outer membrane is polysaccharides, which contain both lipid and carbohydrate, and consist of three parts: (1) lipid A (2) the core polysaccharide and (3) the O side chain. Lipid A region contains glucosamine sugar derivatives, each with three fatty acids and phosphate or pyro phosphate attached. The core polysaccharide is joined to a lipid A. O side chain or O antigen is a polysaccharide chain extending outward from the core. Although the O side chains are readily recognized by host antibodies, gram negative bacteria thwart host defenses by rapidly changing the nature of their o side chains to avoid detection. Lipid A is a major constituents of the outer membrane and LPS helps stabilize the membrane structure. Lipid A often is toxic; as a result the LPS act as an endotoxin and cause some of the symptoms arise in gram negative bacterial infections.

38. Structure of flagella

39. Flagella The word flagellum is the Latin word for whip. It is a thread like loco motor appendages extending outward from the plasma membrane and cell wall and is responsible for swimming motility. They are slender, rigid structures about 20 nm across and up to 15 or 20μm long. Flagellar movement propels the cells at velocities of approximately 20 to 200μm/sec. Examples of bacterial flagella arrangement schemes. A- Monotrichous ;(a single polar flagellum e.g. V.cholerae ) B- Lophotrichous ; ( a cluster of polar flagella e.g Spirillum , Psudomonas fluorescens )  C- Amphitrichous ; (flagella at both the ends either singly or in cluster Aquaspirillum serpens )  D- Peritrichous ; ( cell surface evenly surrounded by several lateral flagella e.g. Proteus vulgaris , Solmonella typhi ) E- Cephalotrichous (two or more flagella at one end of bacterial cell e.g Pseudomonas ) 

40. Flagellar arrange ment

41. Gram negative Four rings present L – with lipopolysaccharides P – peptidoglycan layer M– embedded in the plasma membrane S– plasma membrane rotary engine made up of protein-Mot complex 20 – 30 genes for flagellin 10 gene for hook and basal body protein.

42. Gram positive Two rings are presents One in the peptidoglycan Next in the plasma membrane. Excellent example for self assembly. Flagellar motor driven by protomotive force. cGMP govern the direction in which it rotates.

43. Flagellar motility

44. The filament is in the shape of the rigid helix, and the bacterium moves when this helix rotates. Bacteria having the polar flagella swim in a back and forth fashion; they reverse their direction of swimming by reversing the direction of flagellar rotation. Bacteria having lateral flagella operate in a synchrony to form a bundle that extends behind the cell. However when the flagellar motor reverse, conformational changes occur along the flagella, the bundle flies apart, and the cells tumbles widely. Swimming motility without flagella: Certain helical bacteria (spirochetes) exhibit swimming motility, particularly in viscous media yet they lack external flagella. Gliding movement: Some bacteria such as the species of cyanobacteria (e.g. Cytophaga) and Mycoplasma show gliding movement when come in contact with a solid surface

45. Fimbriae and pili Fimbriae Fine hair like appendages thinner and stiffer than flagella. 1000 in numbers Responsible for more than attachment, twitching motility and jerky motions Pili 1-10 Larger than fimbriae hair like structure. Involved in the conjugation

46. Bacteria can reproduce sexually by conjugation or asexually by binary fission .

47. Staining Simple staining – used single stain. Positive stain – Color cells viewed under colors less back ground. Negative stain - Colorless cell can be viewed under colored background. Differential staining – used more than one staining solution

48. Gram staining The Gram stain , which divides most clinically significant bacteria into two main groups, is the first step in bacterial identification.  Bacteria stained purple are Gram + - their cell walls have thick petidoglycan and teichoic acid. Bacteria stained pink are Gram – their cell walls have thin peptidoglycan and lipopolysaccharides with no teichoic acid.

49. In Gram-positive bacteria, the purple crystal violet stain is trapped by the layer of peptidoglycan which forms the outer layer of the cell. In Gram-negative bacteria, the outer membrane of lipopolysaccharides prevents the stain from reaching the peptidoglycan layer . The outer membrane is then permeabilized by acetone treatment, and the pink safranin counterstain is trapped by the peptidoglycan layer.

50. The Gram stain has four steps: 1. crystal violet, the primary stain : followed by 2. Grams iodine , which acts as a mordant by forming a crystal violet-iodine complex, then 3. alcohol , which decolorizes , followed by 4. safranin , the counterstain .

51. Is this gram stain positive or negative? Identify the bacteria.

52. Acid fast stain( Ziehl – Neelsen’s method) Acid fastness is ascribed by means of high content and variety of lipids, fatty acids and higher alcohols. A lipid in acid fast bacilli is a high molecular weight hydroxy acid wax containing carboxyl group known as mycolic acid. The smear is stained by a carbol fuchsin with the application of heat. Decolourised with 20% sulphuric acid and 98 % alcohol and then counter stained with methylene blue. The acid fast bacteria retain the fuchsin (red) color, while the others take the counter stain. Eg Mycobacteria which causes leprosy and tubercular bacilli are highly resist decolourisation with acid.

53. Results Acid fast:  Bright red  to  intensive purple (B) , Red, straight or slightly curved rods, occurring singly or in small groups, may appear beaded Non-acid fast:  Blue color (A)

54. Capsule staining The capsule stain uses two reagents. Primary Stain : 1% aqueous solution of  Crystal Violet  is used as primary stain. It is applied to a non–heat-fixed smear. Both the cell and the capsular material will take on the dark color. Decolorizing Agent : In the capsule staining, 20%  Copper Sulfate (20%)  is used as a decolorizing agent rather than water. Since the capsule is non-ionic, the primary stain adheres to the capsule but does not bind to it. The copper sulfate solution wash out the primary stain from the capsular material without removing the stain bound to the cell wall. And now the decolorized capsule absorbs the copper sulfate, acquires its blue color and will now appear blue in contrast to the deep purple color of the cell wall.

55. Procedure for capsule staining Take a clean grease free glass slide. Place several drops of crystal violet stain on the slide. Using aseptic technique, add three loopfuls of a culture to the stain and prepare a smear by  gently  mixing with the inoculating loop. With another clean glass slide, spread the mixture over the entire surface of the slide to create a very thin smear. Let the slide stand for 5 to 7 minutes. Allow the smears to air-dry.  (but do not heat fix) Wash smears with 20% copper sulfate solution. Gently   blot dry and examine under oil immersion. Repeat Steps 1–5 for each of the remaining test cultures Record the observations; Indicate the color of the capsule and of the cell on each preparation Result interpretation: The capsule appears as blue colored between deep purple background and cell wall.   Leuconostoc mesenteroides ,  and  Klebsiella aerogenes =  capsule present

56. Gram negative and capsulated:  Neisseria meningitides, Klebsiella pneumoniae, Haemophilus influenza  type B,  Pseudomonas aeruginosa,  Escherichia coli  (few strains),  Yersania pestis , Gram positive and capsulated:   Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogens , Bacillus megaterium , Bacillus anthracis, Capsulated yeast:   Cryptococcus neoformans

57. Structure internal to the cell wall Plasma membrane Cytoplasm Nuclear body Plasmids Inclusion bodies Ribosomes Mesosomes

58. Plasma membrane Thin structure , consists of phospholipids, enclosing the cell. The most accepted model for membrane structure is the fluid mosaic model. Two types of membrane proteins Peripheral protein Intergral protein Hopanoids - steroid like molecules. Often contain mesosomes Acts as a selective barrier.

59. Cytoplasm Cytoplasm in which nucleoid , ribosomes and inclusion bodies are suspended. It consists 80% water ,proteins, carbohydrates lipids, inorganic ions and many low molecular weight compounds. Thick , aqueous, semitransparent and elastic. Nucleoid A single circle of double stranded deoxy ribonucleic acid. 60% DNA , 30% RNA and proteins 10%. Bacteria often contains small circular double stranded DNA molecules called plasmid. Plasmid can replicate autonomously. Types of plasmid – Conjugative plasmid and F factor, Bactereocin encoding plasmid, col plasmid (colcin), virulence plasmid and metabolic plasmid.

60. Ribosomes esosomes Protein synthesis. provide granular appearance for cytoplasms

61. Ribosomes Protein synthesis Granular appearance Prokaryotic ribosomes are 70S.(50S+30S) Streptomycin and gentamicin interfere with 30S subunits Erythromicin and chloramphenicol interfere with 50 S subunits. Mesosomes – folded invaginations in the plasma membrane.

62. Inclusion bodies ( reserve deposits of food) Organic inclusion bodies Glycogen and starch PHB(poly β - hydroxy butyrate) Cyanophycin granules Carboxysomes Gas vacuoles and gas vesicles Poly saccharide granules Lipid inclusions Sulfur granules In organic inclusion bodies Poly phosphate granules or volutin Metachromatic granules Magnetosomes

63. Endospore Bacteria can survive unfavorable conditions by producing an endospore .

64. Endospore formation Metabolically dormant structures Endospore (i.e. produced with in the cell) or exospores (i.e. produced external to the cell). e.g. Bacillus and Clostridium, Sporosarcina Resistant to the environmental stresses such as heat, ultraviolet radiation, gamma radiation, chemical disinfectant and desiccation. Endospores are remain viable for over 100,000 years Unusual resistance in spores in mainly due to the presence of dipicolinic acid, in combination with calcium ions. Outer coat which are protein in composition with high cysteine content and resemble keratin. Reason for heat resistant lie in the fact that dehydration of cytoplasm. Tough protein like spore coat help to protect spore core from harmful effects of chemicals.

65. Stage 1. Axial filament formation - In the first stage the vegetative cell replicates its nucleic acid to form two complete copies of DNA. The nucleic acid extends as a continuous filament along the entire length of the cell. Stage2. Formation of septum The second stage of different endospore differentiation begins with an imagination of the plasma membrane to form a spore septum Stage 3. Forespore development During the next stage of spore formation, a recognizable forespore develops from one of the compartments. Stage 4 . Engulfment of forespore and cortex formation - The forespore is engulfed by the mother cell and a thick cortical layer (the cortex), composed of peptidoglycan is formed. At this stage the forespore becomes increasingly refractile . Calcium dipicolinate is synthesized and incorporated in to the core.

66. Stage 5. Coat synthesis - The fifth stage of spore formation begins with the formation of a spore coat around the spore. The proteins of the coat are different from those synthesized by vegetative cells and probably contribute to the coat’s hydrophobic nature. Stage 6. Completion of coat synthesis - The completed endospore consists of spore coat, cortex, spore wall, and core in association with calcium dipicolinate . Which will be providing refractility and resistant to heat. This envelope, called the exosporium , is composed mainly of protein, carbohydrate and lipid. Stage 7. Lysis of sporangium and spore liberation - Following maturation , the intact spore is released from the cell through lysis of the sporangium. The sporangium is the cell that contains the endospore.

67. Endospore formation

68. Spore germination and outgrowth Specific germination stimulant(L- alanine , glucose) By physical processes (beads) By subleathal effect(60 °C for one hour) Parameters of heat resistance ( i.e time and temperature relationship). D- value (DRT- decimal reduction time) is the time in minutes required to destroy 90% of the population of the cells. The F value is the process describing units expressed in terms of minutes at 121 ° C or a corresponding time temperature relationship to produce the same complete spore killing effect. Z-value is the increase in temperature ( ° C) to reduce the D-value to one tenth. Endospore staining The Schaeffer - Fulton Stain The two genera which we will study are: Clostridium is an anaerobic organism that forms spores. Tetanus, botulism, gas gangrene and pseudomembranous colitis are diseases caused by different species in this genus. Bacillus is a common aerobic genus whose species can form endospores . Anthrax and Bacillus cereus food poisoning are two diseases caused by members of this genus. Spores are extremely resistant structures, difficult to destroy with heat or other physical and chemical disinfecting agents.  

69. EXPERIMENT   Prepare a smear of Bacillus megaterium , allow the smear to dry and then heat-fix. Place the slide on the staining rack in the sink and flood the smear with malachite green stain. Heat the stain to steaming by passing a lit bunsen burner over the smear.  Don't overheat the stain!  Once the steaming stops, pass the bunsen burner over the slide again.  As the stain evaporates add more stain.  Continue this procedure for 5-10 minutes. Wash the smear gently and thoroughly with running water. Counterstain with aqueous safranin for 1 minute. Wash the slide with water, blot gently and allow the smear to air dry. Observe under oil immersion

70. Bacterial reproduction and growth kinetics Multiplication and division cycle Binary fission Gram negative cell- constriction followed by membrane fusion Gram positive cell –formation of cross wall Population growth Growth on solid surface Growth in liquid Liquid batch culture (Closed) Growth in open culture Growth and genetic exchange Transformation Transduction Conjugation

71. Population growth In binary fission one cell divides, producing two cells. Thus, if we start with a single bacterium, the increase in population is by geometrical progression. 1 → 2 →2 2 →2 3 →2 4 →2 5 …2 n . Where n is the number of generations. Each succeeding generation, assuming no cell death, doubles the population. The total population N at the end of the given time period would be expressed N = 1 X 2 n Initial population of N cells as distinct from one cell, at the ‘ n’th generation the population will be N= N X 2 n Where N is the final cell number , N is the initial cell number n the number of generation. To express the equation in terms of n , then Log N =Log N + n log 2 Log N -log N = n log 2

72. Log N -log N Log N -log N n = ---------------- = ------------------------- log 2 0.301 n = 3.3 ( Log N -log N ) Time interval between one cell division and next generation is called the generation time . actual generation time = n/t t= hours or minutes of exponential growth

73. Bacterial growth curve at batch culture Lag phase Log phase ( tropho phase –Primary metabolites) – eg ethanol, lactic acid, and certain aminoacid ) Stationary phase ( idio phase – secondary metabolites synthesized – antibiotics, nucleocides , and quinolines ) Death phase Transition phase

74. Stages in the Normal Growth Curve Data from an entire growth period typically produce a curve with a series of phases Lag Phase Exponential Growth Phase Stationary Growth Phase Death Phase

75. Continuous culture of micro organism A system with constant environmental conditions maintained through continual provision of nutrients and removal of wastes. Two major type of continuous culture systems 1. chemostat 2. turbidostat Chemostat : the culture medium for the chemostat possesses an essential nutrient in limiting quantities. Because one nutrient is limiting the growth rate is determined by the rate at which a new medium is fed in to the growth chamber, and the final density depends on the concentration of the limiting nutrients. The rate of nutrient exchange is expressed as D = f/V If the dilution rate rises too high the microorganism can actually be washed out of the culture vessel before reproducing because the dilution rate is greater than the removal rate . Turbidostat: A photo cell that measures the absorbance or turbidity of the cell in a growth vessel. The flow rate of the medium through vessel is automatically regulated to maintained pre determined turbidity or cell density

76. Environmental factor that effect or influences growth and survival Physico chemical factors Temperature Minimum – no growth Optimum- more rapid Maximum – no growth When temperature raises chemical and enzymatic reaction proceed more rapidly till optimum rate achieved. Beyond this certain protein may become irreversibly damaged- thermal lysis may leads to loss of viability. pH Optimum pH 7.4 and 7.6 . Sub optimal 5 – 8.5 Lactobacilli at vaginal vaults prevents the growth of many opportunist pathogen Acidophiles , neutrophiles and alkalophiles

77. water activity and solute Water activity (Aw) is defined as the vapor pressure of water in the space above the material relative to the vapor pressure above pure water at the same temperature and pressure. Ratio of the solution vapor pressure to that of pure water Availability of oxygen Important for its metabolism. Strongly oxygen dependent bacteria grow as a thin pellicle on the surface where the oxygen is more available. The inability of the oxygen to diffuse inadequately into a fluid culture is the factor which causes onset of stationary phase. Nutrition and growth Bacteria for its nutritional requirements needs carbon, nitrogen, water, phosphorous, potassium and sulphur with minor requirements for trace elements such as magnesium, calcium and iron etc. Chemo lithotroph – utilizes carbon di oxide as a carbon sources. Organic sugars from benzene paraffin waxes and proteins Nitrogen from ammonium ions and deamination of amino acids.

78. Isolation of pure culture Spread Plate Technique Streak plate techniques Pour plate method

79. Isolation procedures A. Spread Plate Technique In this technique, the number of bacteria per unit volume of sample is reduced by serial dilution before the sample is spread on the surface of an agar plate. 1. Prepare serial dilutions of the broth culture as shown below. Be sure to mix the nutrient broth tubes before each serial transfer. Transfer 0.1 ml of the final three dilutions (10-5, 10-6, 10-7) to each of three nutrient agar plates, and label the plates. 2. Position the beaker of alcohol containing the glass spreader away from the flame. 3. Remove the spreader and very carefully pass it over the flame just once (lab instructor will demonstrate). This will ignite the excess alcohol on the spreader and effectively sterilize it. Spread the 0.1 ml inoculum evenly over the entire surface of one of the nutrient agar plates until the medium no longer appears moist. Return the spreader to the alcohol.

80. Continued ……………….. 4. Repeat the flaming and spreading for each of the remaining two plates. 5. Invert the three plates and incubate at room temperature

81. Petri plates

82. Streak plate techniques The streak plating technique isolates individual bacterial cells (colony-forming units) on the surface of an agar plate using a wire loop. The streaking patterns shown in the figure below result in continuous dilution of the inoculum to give well separated surface colonies. Once again, the idea is to obtain isolated colonies after incubation of the plate. 1. Label two nutrient agar plates No. 1 and No. 2. 2. Prepare two streak plates by following two of the 3 streaking patterns shown in the figure below. Use the 10-1 dilution as inoculum. 3. Invert the plates and incubate at room temperature

83. Multiple Streak plate method

84. Different streaking methods

85. Pour plate method The bacterial culture and liquid agar medium are mixed together. After mixing the medium, the medium containing the culture  poured  into sterilized petridishes ( petriplates ), allowed solidifying and then incubated. After incubation colonies appear on the surface.

86. Isolation method Pour plate method Dilution method

87. Cultural characteristics

88.   Appearance: Many fungi produce colonies with a fluffy appearance similar to cotton wool. The molds produce colonies which on aging develop dry chalky appearance. Size: Colonies ranging from extremely small (pinpoint), measuring only fraction of millimeter in diameter, to large colonies measuring 5 to 10 mm in diameter. Young colonies are smaller than the older colonies; therefore the time at which the measurements are must be stated. Margin or edge: The periphery of the bacterial colonies may take one of the several different patterns, depending on the species. The margin may be entire, undulate, crenate, dentate, lobate , rhizoidal or filamentous.

89. Colony forms: The colony shape may be circular, filamentous, rhizoidal, punctiform , irregular or spindle shape.

90. Surface texture : Depending on the species, the colony surface may be smooth (shiny or glistening; or rough (dull, granular, or matte); or mucoid ( slimmy or gummy). Certain species have colonies possessing a highly wrinkled surface. Pure culture can exhibit surface variation. One of the commonest variations is known as S→R variation. For several species of pathogenic bacteria, the surface texture of colonies may bear a relation to virulence. For instance S colonies of S.pneumoniae or Solmonella species are virulent but R colonies or not. The strains of Mycobacterium tuberculosis a rough surface showing serpentine cords is usually a good indicator of virulence.

91. Elevation: Depending on the species, the colonies may be thin to thick, and the surface may be flat or it may exhibit varying degree of convexity. Consistency: This can be determined by touching a transfer needle to the colony. Optical features: The colony may be transparent or translucent (foggy appearance) or opaque (not permitting light to pass through) or irridiscent (rainbow color). Chromogenesis or pigmentation: Many microbes develop colonies which are pigmented. Such colored substance is either water soluble or insoluble. Some species produce water insoluble pigment thus causing the colonies becomes colored. Some species which forms pigmented colonies are Serratia marcescens – red , Chromobacterium violaceum - violet, and Staphylococcus aureus – golden yellow, Micrococcus luteus – Yellow. Some colonies produce pigments that are water soluble; that diffuse into the surrounding agar and stain it.

92. Pseudomonas aeruginosa forms blue water soluble pigment called pyocyanin . Some pigments are only sparingly water soluble and may precipitate in the medium. E.g. Pseudomonas chlororaphis forms a pigment called chlororaphin which accumulates in the form of green crystals around the colonies. Certain water soluble pigments are fluorescent; i.e., the agar medium around the colonies glows white or blue green when exposed to ultra violet light. For example, P.aeruginosa produces not only the nonfluorscent pigment pyocyanin and also a fluorescent pigment pyoverdin .

93. Bacterial toxins Pathogens which can cause disease under if it presented correct set of conditions are called opportunist pathogen. E.g Staphylococcus epidermis and P. aeruginosa. Pathogens produce toxins. Toxins are products of bacteria that produce immediate host cell damage Toxins are classified in to endo toxin ( cell wall related) and exotoxin (Product released extracellularly as the organism grows). Endo toxin is generally released from lysed or damaged cells and posseses multiple biological properties like induce fever, initiate the complement and blood cascade and stimulate tumour necrosis factor. Exo toxins are calssified in to A-B toxin, cytotoxic toxins and super antigen toxins. A-B toxin mediated enzymatic toxicity , Cyto lytic toxins causing lysis( haemolysins and Phospholipases ) and super antigens stimulate immune response cell to produce cytokines there by massive inflammatory reaction

94. Pure culture preservation and maintenance To maintain pure culture for extended periods in a viable conditions, without any genetic change is referred as Preservation. The aim of preservation is to stop the cell division at a particular stage i.e. to stop microbial growth or at least lower the growth rate. Due to this toxic chemicals are not accumulated and hence viability of microorganisms is not affected.

95. OBJECTIVES OF PRESERVATION To maintain isolated pure cultures for extended periods in a viable conditions. To avoid the contamination. To restrict genetic change (Mutation).

96. METHODS OF PRESERVATION AND MAINTENANCE OF MICROBIAL CULTURE The method of preservation is mainly of two types- SHORT TERM METHODS Periodic transfer to fresh media Preservation of bacteria using glycerol Storage by drying method Storage by refrigeration  LONG TERM METHODS Mineral oil or liquid paraffin storage Storage in saline suspension immersion in distilled water Storage in sterile soil Cryopreservation  Stored in silica gel

97. SHORT TERM METHODS Periodic transfer to fresh media Culture can be maintained by periodically preparing a fresh culture from the previous stock culture. Many of the more common microbes remain viable for several weeks or months on a medium like Nutrient agar. It is an advantageous as it is a simple method and any special apparatus are not required. However it is easy to recover the culture. The transfer has the disadvantage of failing to prevent changes in the characteristics of a strain due to development of variants and mutants and risk of contamination is also more in this process.

98. PRESERVATION OF BACTERIA USING GLYCEROL  Bacteria can be frozen using 15% glycerol. The glycerol is diluted to 30% and an equal amount of glycerol and culture broth are mixed, dispensed into tubes, and then frozen at -10˚ C. The viability of organisms varied such as Escherichia coli, Diplococcus pneumonia etc. viable for 5 months, Haemophilus influnzae viable for 4 months, Neisseria meningtidis for 6 weeks and Neisseria gonorrhoeae for 3 weeks

99. STORAGE BY DRYING METHOD Spores of some microbes which are sensitive to freeze- drying, can be preserved by drying from the liquid state rather than the frozen state. Different procedures of drying methods are as follows: Paper disc: A thick suspension of bacteria is placed on sterile discs of thick absorbent paper, which are then dried over phosphorus pentoxide in a desiccation under vacuum. Gelatin disc: Drops of bacterial suspension in gelatin are placed on sterile plastic Petri plates and then dried off over P2O5 under vacuum. L-drying: Bacteria in small ampoules are dried from the liquid state using a vacuum pump and desiccant and a water bath to control the temperature. In this suspension of the organisms are dried under vacuum from the liquid state without freezing taking place. Apart from the mentioned methods the organisms are also dried over Calcium Chloride in vacuum and are stored in the refrigerator. At such conditions the organisms survive for longer period than the air dried cultures.

100. STORAGE BY REFRIGERATION Culture medium can be successfully stored in refrigerators or cold rooms, when the temperature is maintained at 4˚C. At this temperature range the metabolic activities of microbes slows down greatly and only small quantity of nutrients will be utilized. This method cannot be used for a very long time because toxic products get accumulated which can kill the microbes.

101. Long term method Mineral oil or liquid paraffin storage In this method sterile liquid paraffin is poured over the slant culture of microbes and stored upright at room temperature. Where as cultures can also be maintained by covering agar slants by sterile mineral oil which is stored at room temperature or preferably at 0-5°C. It limit the oxygen access that reduces the microorganism’s metabolism and growth, as well as to cell drying during preservation. The preservation period for bacteria from the genera Azotobacter and Mycobacterium is from 7-10 years, for Bacillus it is 8-12 years

102. STORAGE IN SALINE SUSPENSION Bacterial culture is preserved in 1% salt concentration in screw caped tubes to prevent evaporation. The tubes are stored in room temperature. Whenever needed the transfer is made on Agar Slant.

103.  IMMERSION IN DISTILLED WATER Another inexpensive and low-maintenance method for storing fungal culture is to immerse them in distilled water. Fungi can be stored in this method at 20˚C, survived up to 2-10 years depending upon the species. FOR SPORULATING FUNGI: It involves inoculating agar slants of preferred media with fungal cultures and then incubating them at 25˚C for several weeks to induce sporulation. Sterile distilled water(6-7 ml) is added aseptically to the culture, and the surface of the culture is scraped gently with a pipette to produce a spore and mycelial slurry. This is kept in sterile glass vial at 25˚C and to retrieve a culture, 200-300µl of the suspension is removed from the vial and placed on fresh medium.

104. STORAGE IN STERILE SOIL It is mainly applied for the preservation of sporulating microorganisms. Fusarium , Penicillium , Alternaria , Rhizopus etc. proved successful for store in sterile soil. Soil storage involves inoculation of 1ml of spore suspension into soil (autoclaved twice) and incubating at room temperature for 5-10 days. The initial growth period allows the fungus to use the available moisture and gradually to become dormant. The bottles are then stored at refrigerator. Viability of organisms found around 70-80 years.

105.   Lyophilization (Freeze–drying) It is a vacuum sublimation technique. Freeze drying products are hygroscopic and must be protected from moisture during storage. By freezing the cells in a medium that contain a lyoprotectant (usually sucrose) and then pulling the water out using vacuum(sublimation), cells can be effectively preserved. Freezing must be very rapid, with the temperature lowered to well below 0˚C (as such -20˚C). Lyophilized cultures are stored in the dark 4˚C in refrigerators. Many microbes preserved by this method have remained viable and unchanged in their characteristic more than 20 years. It is very advantageous as only minimal storage space is required to preserve .

106. Cryopreservation  Cryopreservation (i.e. freezing in liquid nitrogen at -196˚C or in the gas phase above the liquid nitrogen at -150˚C) helps survival of pure cultures for long storage time. In this method, the microorganisms of culture are rapidly frozen in liquid nitrogen at -196˚C in the presence of stabilizing agents such as Glycerol or Dimethyl Sulfoxide (DMSO) that prevent the cell damage due to formation of ice crystals and promote cell survival. By this method species can remain viable for 10-30 years without undergoing change in their characteristics.

107. Stored in silica gel Microbes can be stored in silica gel powder at low temperature for a period 1- 2 years. The basic principle in this technique is quick desiccation at low temperature, which allows the cell to remain viable for a long period of time. Some of the species which are preserved on anhydrous silica gel are such as- Saccharomyces cerevisiae, Aspergillus nidulans , Pseudomonas denitrificans , Escherichia coli etc.

108. ADVANTAGES : Removal of water at low temperature Thermolabile materials can be dried. Sterility can be maintained. Reconstitution is easy. DISADVANTAGES Many biological molecules are damaged by the stress associated with freezing, freeze- drying, or both. E.g. the process of drying causes extensive damage to molds, protozoa, and most viruses. Hence, these microorganisms can not be stored by this method. The product is prone to oxidation, due to high porosity and large surface area. Therefore the product should be packed in vacuum or using inert gas. Cost may be an issue, depending on the product.

109. Enumeration of microorganism Direct methods Cell counts Smear counts Comparative Membrane filter count Electronic counters Indirect methods Total volume Turbidimetric methods Chemical methods Dry weight determination

110. Measurement of bacterial growth Total count – both living and dead cells Direct microscopic count ( Petroff - Hausser counting chamber and coulter counter or flow cyto meter. Bacteria / mm 3 =(bacteria/square)(25 squares)(50)(dilutions)CFU Turbidity methods Dry weight determinations Nitrogen protein and nucleic acid determinations Viable count - only the living cells Pour plate (counting colonies on agar) No of colonies X dilution factor =---------- cells /ml Surface spread or surface drop (counting the colonies on the agar) Membrane filter method(colonies growing on membranes on agar surface) Most probable number(counts based on the proportion of liquid cultures growing after receiving low inoculam)

111. Fig. 7.17

112. Turbidity

113. Fig. i7.6

114. Measurement of bacterial growth continued Rapid method (indirect viable count) Epifluorescence (image analysis) Adenosine triphosphate methods (bioluminescence) Impedance methods Manometric methods

115. Culture Media Artificially prepared mixture of various nutrients for the growth and multiplication of micro organisms. A culture media should provide suitable carbon, nitrogen, energy sources and nutrients. Common ingredients in media Water - vehicle , copper distilled water cannot be used because it inhibits bacterial growth. Peptone – partially digested protein obtained from lean meat, heart, muscle, casein, fibrin and soya meal etc. it also acts as buffer it is a hygroscopic and stored in a air tight container. Yeast extract – from baker’s yeast or Saccharomyces . Contains carbohydrates, amino acids and growth factors and inorganic salts. Meat extract – from fresh lean meat extracted from hot water. Contains gelatin, peptones, proteases, amino acids, creatine and creatinine , purines and mineral salts, carbohydrates and growth factors, includes thiamine, nicotinic acid riboflavin, pyridoxine and pantothenic acid. Agar - obtained from seeweeds algae. Agrophtes likew gelidium , gracileria , hyophae , gelidiella etc, It is a mixture of aggarose (70%) and Agaropectin (30%).

116. Properties of agar Obtained from different seeweeds of algae. Good solidifying agent in 2% conc. No nutritional value Bacteriologically inert Resistant to the action of all micro organism. Stable at different tem used for incubation. Melts at 95-98°C and remain liquids up to 40 - 42°C. Gets solidified at 40°C. Easily economical and available.

117. Types of culture media Depending on physical state. Solid (1.5 – 2.0 % agar) Semisolid (0.2- 0.5%agar) Liquid media Depending on chemical composition. Simple or basal media Synthetic or defined media Complex media Depending on oxygen requirement Aerobic media ( MacConkey’s broth) Anaerobic medium (Robertson’s cooked meat medium) Depending on functional type Supportive or general purpose media ( Tryptic soy broth or agar ) Enriched media -addition of substances to fortified the fastidious organism to the basal medium. (blood agar [ Streptococcus], chocholate agar [ Neisseria , Heamophilus ], and Loffler serum agar [ Corynebacterium diptheriae ].

118. Enrichment medium- addition of specific substance which inhibit the growth of unwanted bacteria and allow the growth of wanted bacteria.(e.g. Tetra thionate broth Selenite F broth , inhibits E.coli and allow the pathogenic Shigella and Salmonella Speciesi ). Selective media ( Mannitol salt agar for S. aureus , MacConkey agar for E.coli , deoxy citrate agar for Salmonella and shigella species ) Indicator media(Wilson Blair medium fo r Salmonella typhi which reduces sulphite to sulphide in the presence of glucoseand the colonies has black metallic shine). Differential media ( MacConkey’s medium used to differentiate lactose (identified as red colonies )and non lactose fermentor (white colonies)). Transport medium (Stuarts transport medium , Amies transport medium) Neisseria can be over grown by E.Coli or Shigella and salmonella during transportation. Storage medium ( Dor set’s egg medium, blood agar, nutrient agar stabs and Robertson cooked medium)

119. Anaerobic media Solid anaerobic media To eliminate oxygen dissolved in the culture mediareducing substances areadded in the solid and liquid culture media. Glucose and other reducing sugars , SH- containing aminoacid for e.g. cysteine and other reducing agent sodium thioglycollate . Common anaerobic medium is Reinforced Clostridial Medium. Liquid anaerobic media Small number of organism can be dectected by liquid medium. Robertson’s cooked meat medium containing 1 cm depth of diced lean heart meat in the bottom. Recently heated to 100 °C to expel oxygen or freshly prepared media is used . Thioglycollate medium –contains oxygen reduction indicator.one third of the depth of the medium was coloured . Anaerobic jars- Mc Intosh and Flides jar or Brewer and allgeier Gaspak jar