Microbiology & Infection Prevention.pptx

This is a presentation on the course, Microbiology for health related students who are reading the subject. I can also be useful to student from different fields taking the course.
It contains 144 slides.

1. MICROBIOLOGY AND INFECTION PREVENTION SAMUEL ESSIEN-BAIDOO

2. By the end of the course, students should be able to; Describe the range of microorganisms and their classifications Discuss the role of pathogenic organisms Identify the role of the acquired and innate immune systems Explain the role of infectious organisms in disease causation Discuss infection control practices that prevent spread of infection Demonstrate skills in standard precautions including hand washing, gowning, and gloving in the healthcare environment.

3. Unit 1: Overview of Microbiology

4. Introduction Microbiology is the study of organisms that are too small to be seen by the naked eye. The microbes have coexisted with humans from the beginning of civilization providing both beneficial and detrimental roles to human life. Although not always recognized at the time microbes have dramatically. They are ubiquitous and have many economic importance.

5. History The science of microbiology started with the invention of the microscope. The first reported compound microscope was made by Zacharias Jansen a Dutch spectacle maker. One of the first to use a microscope was Robert Hooke in 1665. He used the microscope to observe cells.

6. Anton Von Leeuwenhoek The first accurate description of microbes was reported in 1674 by Anton von Leeuwenhoek a Dutch lens maker. He was the first person to see and describe living microbes. He observed and described microorganisms as “Animalcules”.

7. Aristotle The belief in the spontaneous generation of life from nonliving matter was introduced by Aristotle, who lived around 350BC. According to Aristotle it was, “ readily observable that aphids arise from the dew which falls on plants, fleas from putrid matter, mice from dirty hay. This belief remained unchallenged for more than 2000 yrs

8. Robert Hooke (1635-1703 AD) Developed compound microscope. Was the 1 st to coin the term ‘cell’

9. GOLDEN ERA ( Louis Pasteur) Father of medical Microbiology. In his famous experiment, he took a Swan Neck Flask and boiled nutrient rich broth inside it. He pointed that no growth took place in the flask as dust and germs had been trapped on the walls of the curved neck.

10. In 1897,he suggested that mild heating at 62.8°C (145°F) for 30 minutes rather than boiling was enough to destroy the undesirable organisms without ruining the taste of product, the process is called Pasteurization . Coined the term ‘microbiology’. Disproved the theory of spontaneous generation. Demonstrated that anthrax was caused by bacteria and proved the vaccine for disease. Developed live attenuated vaccine for the disease

11. ROBERT KOCH (1843-1912) Developed four postulates that aided in the definitive establishment of the germ theory of disease. They are: A specific organism can always be found in association with a given disease. The organism can be isolated and grown in pure culture in laboratory The pure culture will produce disease when inoculated into a susceptible animal It is possible to recover the organism in pure culture from the experimentally infected animal.

12. Edward Jenner(1729-1823) Discovered the technique of vaccination Fanne Eilshemius Hesse(1850-1934) First proposed the use of agar in culture media. It was not attacked by most of the bacteria. Richard Petri(1852-1921) Developed the Petri dish, used for solid culture media

13. Alexander Flemming (1881-1955 AD) Discovered Penicillin from Penicillium notatum that destroys several pathogenic bacteria. Paul Ehrlich(1854-1915 AD) Discovered treatment of Syphilis by using arsenic. Studied toxins and anti toxins in quantitative terms and laid foundations of biological standardizations.

14. Nobel Laureates Year Laureates Contribution 1901 VonBehring Dipthantitox 1902 RonaldRoss Malaria 1905 RobertKoch TB 1908 Metchinkoff Phagocytosis 1945 Flemming Penicilin 1962 WatsonCrick Structure DNA 1968 Holley,Khorana GeneticCode 1997 Pruisner Prions 2002 Brenner,Hervitz Genetic regulation of organ Development and cell health

15. Classification of Microorganisms

16. Taxonomy

17. Taxonomy Domains Kingdom Phylum Class Order Family Genus Species

18. Evolution - living things change gradually over millions of years Changes favoring survival are retained and less beneficial changes are lost All new species originate from preexisting species Closely related organism have similar features because they evolved from common ancestral forms Evolution usually progresses toward greater complexity

19. Three Domains! Eubacteria True bacteria, peptidoglycan Archaea Odd bacteria that live in extreme environments, high salt, heat, etc. (usually called extremophiles) Eukarya Have a nucleus & organelles (humans, animals, plants)

20. Naming Microorganisms Binomial (scientific) nomenclature Gives each microbe 2 names: Genus - noun, always capitalized Species - adjective, lowercase Both italicized or underlined Staphylococcus aureus (S. aureus) Bacillus subtilis (B. subtilis)

21. Species and Subspecies Species Collection of bacterial cells which share an overall similar pattern of traits in contrast to other bacteria whose pattern differs significantly Strain or variety Culture derived from a single parent that differs in structure or metabolism from other cultures of that species. Type Subspecies that can show differences in antigenic makeup (serotype or serovar), susceptibility to bacterial viruses (phage type) and in pathogenicity (pathotype)

22. Classification Basis in the Prokaryotes Growth on media Microscopic morphology Macroscopic morphology – colony appearance Biochemical characteristics Serological analysis Genetic and molecular analysis

23. 1. Growth on Media Suitable criteria for purposes of general bacterial classification -Growth on bacteriologic media. In contrast to viruses and most parasites, many bacterial pathogens can be isolated on solid agar-containing media.

24. 2. Microscopic morphology Microscopic characterization The Gram stain, together with visualization by light microscopy, has been among the most informative methods for classifying the eubacteria. This staining technique broadly divides bacteria on the basis of fundamental differences in the structure of their cell walls

25. 3. Macroscopic morphology – colony appearance C olony morphology on the agar plates are used in the identification Form, Texture, Colours etc. are also used in identification and classification

26. 4. Biochemical Tests Tests such as the Metabolism of CHO, Citrate ; Production of gas, Urease, Oxidase, Catalase, Proteinase; Nitrate reduction etc. These tests are helpful in the biochemical characterization of microorganisms and are used in their classification

27. 5. Serological analysis Immunologic Tests — Serotypes, Serogroups, and Serovars The designation “ sero ” simply indicates the use of antibodies (polyclonal or monoclonal) that react with specific bacterial cell surface structures such as lipopolysaccharide (LPS), flagella, or capsular antigens. The terms “ serotype ,” “ serogroups ,” and “ serovars ” are, for all practical purposes, identical — they all use the specificity of these antibodies to subdivide strains of a particular bacterial species.

28. 6. Genetic and molecular analysis The genetic properties of bacteria allow genes to be exchanged among distantly related organisms. Chemical characterization of bacterial genomic DNA reveals a wide range of nucleotide base compositions among different bacterial strains. The guanine + cytosine (G + C) content of closely related bacteria is similar, indicating that genetic relatedness of DNA from similar organisms can be used

29. A more precise method is DNA sequencing. This method has become a routine procedure, and comparison of the DNA sequences of different genes can give a measure of their relatedness. Thus, DNA sequence differences among rapidly diverging genes can be used to ascertain the genetic distance of closely.

30. Nucleic acid-based Taxonomy! Since 1975, developments in nucleic acid isolation, amplification, and sequencing spurred the evolution of nucleic acid – based subtyping systems. These include Plasmid profile analysis; Restriction Endonuclease analysis; Ribotyping; Pulsed field gel electrophoresis; PCR amplification and restriction; endonuclease digestion of specific genes ; and Nucleic acid sequence analysis etc.

31. Plasmid Profiling: Antibiotic resistance genes on Plasmid Plasmid DNA profile of Pseudomonas aeruginosa BC15. Lane 1 = P. aeruginosa BC15 (pBc15); Lane 2 = Transformed E. coli DH5 α pBC15 and Lane 3 = λ Hind III digest molecular weight marker Raja, C.E., & Selvam, G.S. (2009). Plasmid profile and curing analysis of

32. Restriction Endonuclease analysis

33. Restriction Endonuclease analysis

34. Ribosomal RNA (rRNA) Ribosomes have an essential role in protein synthesis for all organisms. Genetic sequence encodings both ribosomal RNAs (rRNA) and proteins (both of which are required to comprise a functional ribosome) have been highly conserved throughout evolution and have diverged more slowly than other chromosomal genes. Comparison of the nucleotide sequence of 16S ribosomal RNA (rRNA) from a range of prokaryotic sources revealed evolutionary relationships among widely divergent organisms

35. Ribotyping

36. Pulsed field gel electrophoresis

37. PCR amplification and restriction endonuclease digestion of specific genes

38. Unit 2: Principal groups of microbes

39. THE MICROBIAL WORLD 15 February 2023 42 Bacteria, Achaea, Eucarya Prokaryotes Viruses, Prions, Viroids The microbial world Organisms Living Algae Protozoa Fungi Helminthes Eukaryotes Infectious agents Non living

40. MICROBIAL WORLD Members of the microbial world consist of two major cell types- simple prokaryotic and the complex eukaryotic. All organisms fall into one of three domains, based on the chemical composition and cell structure. These are the bacteria, Achaea and the Eucarya. 15 February 2023 43

41. BACTERIA They are all single celled prokaryotes. Most have specific shapes; cylindrical or rod-shaped, spherical or round or spiral. Most have rigid cell walls, which are responsible for the shape of the organism. The walls contain an unusual chemical compound called peptidoglycan which is not found in organisms of other domains. 15 February 2023 44

42. Bacteria They multiply by binary fission in which one cell divides into two cells each generally identical to the original. Many can move using appendages extending from the cell called flagella. Bacteria are single-celled prokaryotes that have peptidoglycan in their cell wall. 15 February 2023 45

43. THE PROKARYOTIC CELL 15 February 2023 46

44. Archaea 15 February 2023 47

45. The Eucarya 15 February 2023 48

46. The Eucarya All algae, fungi, protozoa and multicellular parasites considered in this course are eukaryotes. Microbial members of the Eucarya are the algae, fungi, and protozoa. 15 February 2023 49

47. Algae Algae can be single celled or multicellular They can use sunlight as a source of energy Phytoplankton produce oxygen for aquatic life Paralytic shelfish poisoning – Gymnodinium brevi Gunyaulax Brevitoxins , saxitoxins and gaunyautoxin Have rigid cell walls cellulose and pectin 15 February 2023 50

48. Fungi Fungi are saprophytic, parasitic or commensal organisms. The cell wall consists of polysaccharides, polypeptides and chitin. Yeast, moulds or filamentous and dimorphic Yeast- Candida Albicans, Cryptococcus neoformans Moulds are multicellular – Aspergillus species Dimorphic fungi- Blastomyces dermartitidis 15 February 2023 51

49. Protozoa Protozoa are single-celled organisms that are motile by a variety of means. They use organic compounds as food. Giardia lamblia, Trichomonas vaginalis Entamoeba histolytica. Plasmodium and Cryptosporidium parvum. 15 February 2023 52

50. Viruses, Viroids and Prions The non-living members of the microbial world are not composed of cells. They are considered as obligate intracellular parasites and include viruses, viroids and prions. Viruses are a piece of nucleic acid surrounded by a protein coat. They can infect all members of the three domain. 15 February 2023 53

51. VIRUSES Small infectious pathogen composed of one or more nucleic acid molecules usually surrounded by a protein coat." They typically reproduce by latching onto the wall of a cell and inserting their genetic material - i.e., the nucleic acids - into the cell. This genetic material then uses the cell's machinery to make more copies of the virus. Typically, these copies overrun the cell until it bursts. Viruses use a large number of sneaky tricks to overcome the defense mechanisms of the cell. 15 February 2023 54

52. VIRAL DISEASES The common cold ,influenza (the flu) , measles, rubella. Mumps, warts, chickenpox , smallpox, acquired immunodeficiency syndrome (AIDS) , herpes , hepatitis, rabies, poliomyelitis (polio), encephalitis. Yellow fever and many more. 15 February 2023 55

53. VIROIDS Small infectious pathogen composed of a low molecular weight RNA molecule". It has no protein coat. It is a single-stranded circular RNA! Viroids don't function as messenger RNAs, so they don't make the cell synthesize enzymes: they rely completely on pre-existing enzymes in the host for their reproduction. Most known viroids cause diseases in plants. The first viroid was discovered in 1971, by Diener . It's called the potato spindle tuber virus (PSTV), it Makes potatos abnormally long and sometimes cracked. 15 February 2023 56

54. 15 February 2023 57 VIROIDS

55. PRIONS Prions consist only of protein without nucleic acid. They cause severe neurodegenerative diseases in animals Prions are small, proteinaceous infectious particles that contain no detectable nucleic acid of any form, but are transmissible among certain animals, where they cause fatal brain diseases. 15 February 2023 58

56. PRIONS These particles are rod-shaped, 165 nanometers long and 11 nanometers in diameter, and they consist largely of a protein called “PrP Sc ”, having molecular weight 33,000-35,000. They are able to resist inactivation by boiling, acid (pH 3-7), ultraviolet radiation (254 nm), formaldehyde, and nucleases! They can be inactivated by boiling in detergents, alkali (pH > 10), autoclaving at 132 degrees centigrade for over 2 hours, and denaturing organic solvents such as phenol. 15 February 2023 59

57. PRIONS Prions are a modified form of a protein naturally occurring in the brain ( PrP ) and spread by auto catalyzed chain reaction. Prions cause "mad cow disease“ known as bovine spongiform encephalopathy. This disease infected thousands of cattle in England, Starting in the mid-1980s because they were being fed offal containing nerve tissue from sheep infected with a prion-caused disease called "scrapie". 15 February 2023 60

58. PRIONS There are a number of prion-induced brain diseases in people, such as Creutzfeldt-Jakob disease (which occurs spontaneously in about one in a million people) and FFI There are also prion-induced brain diseases in mink, cats, deer and moose. 15 February 2023 61

59. Bacteria Overview of Clinically Relevant Bacteria

60. Classification of Bacteria Morphology – shape, color, gram specificity Metabolism Molecular techniques – Forensics, DNA fingerprints, RNA, protein analysis

61. Classification based upon anatomical features 3 common shapes Some unusual shapes also:

62. Classification based upon anatomical features Other unusual bacteria Spirochetes Cell wall-less Stalked Filamentous Myxobacteria fruiting bodies Streptomyces

63. Classification based upon staining Gram Positive vs Gram Negative Hans Christian Gram -- 1884 -- Crystal violet Gram positive structure -- thick layer of peptidoglycan Gram negative structure -- inner vs outer membranes -- lipopolysaccharides and endotoxins Acid fast staining -- Mycobacterium

64. Classification based upon metabolism Heterotrophic Autotrophic Photosynthetic bacteria -- cyanobacteria -- purple sulfur bacteria Chemoautotrophic ‘Metabolically defective’ Rickettsia Chlamydia Rocky Mountain Rocky Mountain Spotted fever wood tick R. rickettsia Image from (and good source for more about Chlamydia) http://www.chlamydiae.com/docs/biology/biol_devcycle.asp

65. Classification based upon growth requirements Oxygen aerobic anaerobic facultative strict pH acidophilus neutrophilus alkalinophiles Temperature Exogenous (somewhere in the environment). Endogenous (i.e., on or within the human body) Classification based upon Environmental reservoirs are generally divided into those that are

66. 1 Gram Negative Spiral Bacteria Slender and flexible, come in a lot of different shapes More rigid than spirochetes Ex. – Campylobacter jejuni Symptom – tenesmus: the sensation of desire to defecate, which is common and occurs frequently , with out the production of significant amounts of feces (often small amounts of mucous or blood are alone passed).

67. 2 Gram Negative Spirochetes pathogenic very flexible tightly coiled, helically coiled Example syphilis Treponema pallidum

68. 2. Gram Negative Spirochetes Most of pathogenic Very flexible Tightly coiled, helically coiled Example Lyme disease Borrelia burgdorferi (organism gets lodged in tissues)

69. 3 Gram Negative Aerobic Rods Legionella pneumophila Lower respiratory tract infection Needs oxygen

70. Gram Negative Aerobic Rods Bordetella pertussis – whooping cough Needs oxygen

71. Gram Negative Aerobic Rods Pseudomonas aeruginosa (pigmented) Needs moisture Common in hospitals Opportunistic pathogen – causes UTI, skin, and lung infection

72. 4 Gram Negative Facultative Rods Vibrio V. cholerae Most well known of group Very severe dysentery. Can lose 10-15 liters of water/day. Leads to hypovolemia – low water, hardly any water in body V. vulnificus Very pathogenic Can cause flesh eating disease, if it gets in a wound V. parahaemolyticus Found in shellfish – oysters Halophile – loves salt (will find in oceans, estuaries) Self limiting

73. Gram Negative Facultative Rods Salmonella Shigella E. coli (0157H7) Enteric

74. 5 Gram Negative Anaerobic Rods Fusobacterium Live in between teeth and gums Cause tooth abscesses and periodontal disease Teeth have nothing to anchor – bone is destroyed

75. 6 Gram Negative Cocci or Coccobaccilli (plump rods) Neisseria gonorrhoeae - sexually a diplococcus Sexually Transmitted Disease very antibiotic resistant

76. Gram Negative Cocci or Coccobaccilli (plump rods) Neisseria meningitidis very infectious and communicable.

77. Gram Negative Cocci or Coccobaccilli (plump rods) Acinetobacter baumanni iv. lwoffi Opportunistic, UTI, skin, and upper respiratory

78. 7 Chlamydia Gram Negative Rods (Transitional) Very short little rods Gram negative Transitional – doesn’t hold stain well Do not have the ability to synthesize own ATP, therefore and obligate intracellular parasite of other animals (humans) Can go asymptomatic for a long time Ex. C. trachomatis – STD, causes eye infection C. psittaci – parrot (associated with birds)

79. 8 Rickettsia Gram Negative Rod (Transitional) Small gram-negative rods Transitional – doesn’t hold stain well Can’t synthesize its own NAD, coenzyme A, therefore an obligate intracellular parasite Causative agent of Rocky Mountain Spotted Fever Example R. Prowazekii

80. 9 Mycoplasma Gram Positive (Transitional ) Gram positive – only because they take in dye in cell membrane, but it washes away Transitional – doesn’t hold stain well. Have no cell wall Can not treat with penicillin Ex. Mycoplasma pneumoniae – causes LRTI Ureaplasma urealyticum – causes UTI Both imbed themselves in the tissue. The most cell damage is done by the immune system destroying the tissue.

81. 10 Gram Positive Cocci Staphylococcus aureus MRSA These bacteria can break down all tissues of body.

82. Gram Positive Cocci Streptococcus pyogenes – no antibiotic resistance right now These bacteria can breakdown all tissues of body.

83. 11 Gram positive Endospore Forming Rods Difficult to get rid of because of endospores Example Clostridium tetani

84. Gram positive Endospore Forming Rods Difficult to get rid of because of endospores Example C. perfringens – gangrene

85. Gram positive Endospore Forming Rods Difficult to get rid of because of endospores Common in hospitals Example C. difficile antibiotic associated pseudomembranous enterocolitis

86. Gram positive Endospore Forming Rods Bacillus B. anthracis – anthrax zoonosis

87. Gram positive Endospore Forming Rods Bacillus B. cereus – food poising Especially in high carbohydrate foods – rice, vermicelli B. thuringiensis – natures insecticide

88. 12 Coryneforms Pleomorphic (many shapes) Example Corynebacterium diphtheriae

89. 13 Mycobacteria Gram positive and Acid Fast Mycobacterium tuberculosis Respiratory Pathogen MDR-TB In the 1950s we sent people with TB to the sanitariums

90. Mycobacteria Gram positive and Acid Fast M. avium intracellular complex (MAC) Really bad bug Currently no drugs can cure it Especially bad for people with AIDS Can cause atypical TB

91. Mycobacteria Gram positive and Acid Fast M. leprae Causative agent of leprosy Not very common Only affects areas of body that are below body temperature Natural reservoir is the armadillo

92. Identification of microbes Unit 3:

93. Introduction The most basic reason that cells are identified/stained is to enhance visualization of the cell or certain cellular components under a microscope. Cells may also be stained to highlight metabolic processes or to differentiate between live and dead cells in a sample. Cells may also be enumerated by microcopy, staining cells to determine biomass in an environment of interest. Cell staining techniques and preparation depend on the type of stain and analysis used. One or more of the following procedures may be required to prepare a sample.

94. Staining

95. Techniques PERMEABILIZATION- treatment of cells, generally with a mild surfactant, which dissolves cell membranes in order to allow larger dye molecules to enter inside the cell. FIXATION-serves to "fix" or preserve cell or tissue morphology through the preparation process. MOUNTING-involves attaching samples to a glass microscope slide for observation and analysis STAINING- application of stain to a sample to color cells, tissues, components, or metabolic processes.

96. GRAM'S STAINING The Gram staining method, named after the Danish bacteriologist who originally devised it in 1844, Hans Christian Gram, is one of the most important staining techniques in microbiology The primary stain of the Gram's Method is crystal violet. Crystal violet is sometimes substituted with methylene blue. The microorganisms that retain the crystal violet-iodine complex appear purple brown under microscopic examination. These microorganisms that are stained by the Gram's Method are commonly classified as gram positive or gram non-negative. Others that are not stained by crystal violet are referred to as gram negative.

97. In Gram's Method, which is based on the ability of a cell in retaining the crystal violet dye during solvent treatment, it is the difference in the microbial cell wall that is amplified. The cell walls for gram-negative microorganisms have higher lipid content than gram-positive cells. Originally, both kinds of cells are penetrated by the crystal violet. Iodine is subsequently added as a mordant to form the crystal violet-iodine complex so that the dye cannot be removed too easily. This step is commonly referred to as fixing the dye. However, the subsequent treatment with the decolorizer, which is a mixed solvent of ethanol and acetone, dissolves the lipid layer from the gram-negative cells.

98. ACID FAST STAINING Acid fast staining is another important differential staining procedure. It is also known as the Ziehl-Neelsen method. This method is used to identify Mycobacterium tuberculosis and M. leprae, the pathogens responsible for tuberculosis and leprosy, respectively. A few species, particularly those in the genus Mycobacterium do not bind simple stains readily and must be stained by a harsher treatment: heating with a mixture of basic fuchsin and phenol (the Ziehl-Neelsen method). Once basic fuchsin has penetrated with the aid of heat and phenol, acid-fast cells are not easily decolorized by an acid-alcohol wash and hence remain red.

99. Non-acid-fast bacteria are decolorized by acid-alcohol and thus are stained blue by methylene blue counterstain. After decolorization the slide may be counter stained with methylene blue or brilliant green. The “acid fast” organisms will appear red while non-“acid fast” organisms will appear blue or green.

100. Specimen Collection Organisms being stained by an acid-fast method are usually taken from a solid or a liquid medium on (in) which they have been cultured from their original source. An aqueous suspension is made (in the case of a solid medium) by taking a small amount of the material and suspending it in a drop of distilled water on a microscope slide. Care should be taken not to make the smear too thick. In the case of a liquid medium, a drop is used directly from the culture container. However, due to the solids from the medium, this method is not always satisfactory. The suspension made by either method is air dried and then “fixed” by passing it rapidly through a Bunsen burner flame two or three times. Allow the smear to cool before staining.

101. Procedure

102. CULTURE MEDIA & CULTURE METHODS

103. Overview Bacteria have to be grown (cultured) for them to be identified. By appropriate procedures they have to be grown separately (isolated) on culture media and obtained as pure for study. History The original media used by Louis Pasteur – urine or meat broth Liquid medium – diffuse growth Solid medium – discrete colonies.

104. Colony Macroscopically visible collection of millions of bacteria originating from a single bacterial cell. Cooked cut potato by Robert Koch – earliest solid medium Gelatin – not satisfactory - liquefy at 24 o C

105. Agar Frau Hesse Used for preparing solid medium Obtained from seaweeds. No nutritive value Not affected by the growth of the bacteria. Melts at 98 o C & sets at 42 o C 2% agar is employed in solid medium

106. Types of culture media Based on their consistency a) solid medium b) liquid medium c) semi solid medium Based on the constituents/ ingredients a) simple medium b) complex medium c) synthetic or defined medium d) Special media

107. Special media Enriched media Enrichment media Selective media Indicator media Differential media Sugar media Transport media Media for biochemical reactions Based on Oxygen requirement - Aerobic media - Anaerobic media

108. Solid media – contains 2% agar Colony morphology, pigmentation, hemolysis can be appreciated. Eg: Nutrient agar, Blood agar Liquid media – no agar. For inoculum preparation, Blood culture, for the isolation of pathogens from a mixture. Eg: Nutrient broth Semi solid medium – 0.5% agar. Eg: Motility medium

109. Complex media Media other than basal media. They have added ingredients. Provide special nutrients Synthetic or defined media Media prepared from pure chemical substances and its exact composition is known Eg : peptone water – 1% peptone + 0.5% NaCl in water

110. Enriched media Substances like blood, serum, egg are added to the basal medium. Used to grow bacteria that are exacting in their nutritional needs. Eg : Blood agar, Chocolate agar

111. Blood agar Chocolate agar

112. Enrichment media Liquid media used to isolate pathogens from a mixed culture. Media is incorporated with inhibitory substances to suppress the unwanted organism. Eg: Selenite F Broth – for the isolation of Salmonella, Shigella Alkaline Peptone Water – for Vibrio cholerae

113. Selective media The inhibitory substance is added to a solid media. Eg : Mac Conkey’s medium for gram negative bacteria TCBS – for V.cholerae LJ medium – M.tuberculosis Wilson and Blair medium – S.typhi Potassium tellurite medium – Diphtheria bacilli

114. TCBS Mac Conkey’s medium

115. Potassium Tellurite media LJ media

116. Indicator media These media contain an indicator which changes its colour when a bacterium grows in them. Eg : Blood agar Mac Conkey’s medium Christensen’s urease medium

117. Urease medium

118. Lactose fermenters – Pink colonies Non lactose fermenters – colourless colonies

119. Transport media Media used for transporting the samples. Delicate organisms may not survive the time taken for transporting the specimen without a transport media. Eg : Stuart’s medium – non nutrient soft agar gel containing a reducing agent Buffered glycerol saline – enteric bacilli

120. Anaerobic media These media are used to grow anaerobic organisms. Eg : Robertson’s cooked meat medium, Thioglycolate medium.

121. CULTURE METHODS Culture methods employed depend on the purpose for which they are intended. The indications for culture are: To isolate bacteria in pure cultures. To demonstrate their properties. To obtain sufficient growth for the preparation of antigens and for other tests. For bacteriophage & bacteriocin susceptibility. To determine sensitivity to antibiotics. To estimate viable counts. Maintain stock cultures.

122. Culture methods include: Streak culture Lawn culture Stroke culture Stab culture Pour plate method Liquid culture Anaerobic culture methods

123. STREAK CULTURE Used for the isolation of bacteria in pure culture from clinical specimens. Platinum wire or Nichrome wire is used. One loopful of the specimen is transferred onto the surface of a well dried plate. Spread over a small area at the periphery. The inoculum is then distributed thinly over the plate by streaking it with a loop in a series of parallel lines in different segments of the plate. On incubation, separated colonies are obtained over the last series of streaks.

124. LAWN CULTURE Provides a uniform surface growth of the bacterium. Uses For bacteriophage typing. Antibiotic sensitivity testing. In the preparation of bacterial antigens and vaccines. Lawn cultures are prepared by flooding the surface of the plate with a liquid suspension of the bacterium.

125. Antibiotic sensitivity testing

126. STROKE CULTURE Stroke culture is made in tubes containing agar slope / slant. Uses Provide a pure growth of bacterium for slide agglutination and other diagnostic tests.

127. STAB CULTURE Prepared by puncturing a suitable medium – gelatin or glucose agar with a long, straight, charged wire. Uses Demonstration of gelatin liquefaction. Oxygen requirements of the bacterium under study. Maintenance of stoke cultures.

128. Gelatin liquefaction Oxidation – Fermentation medium

129. POUR PLATE CULTURE Liquid cultures are inoculated by touching with a charged loop or by adding the inoculum with pipettes or syringes. Uses Blood culture Sterility tests Continuous culture methods Disadvantage It does not provide a pure culture from mixed inocula .

130. Blood culture bottles