Clinical Bacteriology Supplemental Lecture Module 1 PDF

1st Virtual Meeting – 11 Aug. 2020 Clinical Bacteriology Online Class Orientation Supplemental Lecture- Part 1 Reminders: 1. Fill-up your Student information Sheet thru your Section Leader. 2. Study the ass...

1st Virtual Meeting – 11 Aug. 2020 Clinical Bacteriology Online Class Orientation Supplemental Lecture- Part 1 Reminders: 1. Fill-up your Student information Sheet thru your Section Leader. 2. Study the assigned Modules before virtual meeting. 3. Check schedule of the quiz. 4. Don’t be Absent or Late during the virtual meeting Prepared by: RSCRUZ Centro Escolar University- Manila 1  A Microbiology advanced biology course of Biology (study of living organism) Micro means very small- anything so small that it must be viewed with a microscope. Microbiology is the study of very small living organism called as microorganism or microbes (ubiquitous- virtually everywhere). The various categories of microorganism include viruses, bacteria, archaeans, certain algae, protozoa, and certain fungi. Prepared by: RSCRUZ Centro Escolar University- Manila 2  Importance of Medical Microbiology Cultivation/ Propagation of organisms from patient specimen Study of technique for isolation, cultivation and identification of pathogens Study of microbial physiology, growth of requirements, morphology, biochemical, molecular characteristics for diagnosis and treatment of diseases (Antimicrobial susceptibility test) Prepared by: RSCRUZ Centro Escolar University- Manila 3  Microbial Taxonomy Realized phylogeny ( evolutionary history of organism) 3 distinct disciplines a. Classification- Organization of microorganism that share similar morphologic, physiologic, and genetic traits into specific groups or taxa. b. Nomenclature - naming of organism according to established rules and guidelines for universal recognition , with a binomial system ( genus and species) c. Identification-process by which a microorganism’s key features are delineated, which can be compared with other characterized microorganisms Supplemental readings: Chapter 1 p 1-4 -BAILEY & SCOTT’S DIAGNOSTIC MICROBIOLOGY. 12 Ed. Forbes,BA, Sahm, DF, Weissfeld, AS. Prepared by: RSCRUZ Centro Escolar University- Manila 4  Taxonomy Classification/taxonomy Nomenclature Identification Prepared by: RSCRUZ Centro Escolar University- Manila 5  The Classification of Organisms Classification - multilevel grouping of individuals – organisms first classified by Aristotle over 2,000 years ago – Classification scheme of the Middle Ages (polynomial system) was replaced with a binomial system by Linnaeus about 250 years ago. – polynomial - strings of Latin words and phrases containing up to 12 words – binomial - two-part name for each species Prepared by: RSCRUZ Centro Escolar University- Manila 6 Classification by Phenotypic and Genotypic Characteristics Subdivision of subspecies based on : ❑phenotypic differences (abbreviated “subsp.”) Serovarieties based on: ❑ serologic differences (abbreviated “serovar.”) Biovarieties, based on : ❑Biochemical test result differences (abbreviated “biovar”) Prepared by: RSCRUZ Centro Escolar University- Manila 7  Nomenclature The family name is capitalized and has an –aceae ending. e.g., Micrococcaceae The genus is capitalized and followed by the species epithet, which begins with a small letter; both genus and species should be italicized in print but underlined when written in script. e.g., Staphylococcus aureus or Staphylococcus aureus Prepared by: RSCRUZ Centro Escolar University- Manila 8  Schemes of Classification Basis of cell organization, cells are classify into: Five Kingdom Three Kingdom Two Kingdom ( Domain) Prepared by: RSCRUZ Centro Escolar University- Manila 9  The Kingdoms of Life Most biologists use a six-kingdom system. Animalia Plantae Fungi Protista Archaebacteria Bacteria Domains - taxonomic level above kingdoms Prepared by: RSCRUZ Centro Escolar University- Manila 10  Classification of Organisms Clasification of Organisms: 1. Whittaker's Classification(1969) was devised by Robert H. Whittaker based on the nutritional pattern of microorganism: Kingdom Mode of Procuring Food/ Nourishment 1. Animalia- Ingestive 2. Mycetae- Absorptive 3. Plantae- Photosynthetic 4. Protista- unicellular,plant-like, animal-like and fungi-like organism 5. Monera- bacteria and cyanobacteria 2.Marguelles.et.al (1978) came up with the modified classification scheme based on the type of cells: Superkingdom: Eucaryotae which include: Kingdom: 1. Plantae 2. Mycetae 3. Animalia 4. Protista Superkingdom: Procaryotae which includes: Kingdom: 1. Monera Prepared by: RSCRUZ Centro Escolar University- Manila 11 Prepared by: RSCRUZ Centro Escolar University- Manila 12  Understanding the Concept: 1. List down different species of plants, animals, fungi, algae, protozoa and bacteria. 2. Classify them into 2 Kingdom Classification Prepared by: RSCRUZ Centro Escolar University- Manila 13  Groups of Microorganisms : 1.Algae - simple organisms, unicellular, others are aggregation of similar cell with little or no differentiation in complex structure or function. 2.Viruses - very small non-cellular parasite or pathogens of plants, animals and bacteria, even protist. - visualized only with the used of electron microscope. can be cultivated only in living cells. 3.Bacteria - minute, unicellular procaryotic orgnanism, multiply by binary fission, plant-like, microscopic organism which lacks chlorophyll. 4.Protozoa - unicellular eukaryoitc organism, differentiated on the basis of morphology, physiology and nutrition. 5.Fungi - eukaryoticf lower plants devoid of chlorophyll, usually multicellular but not differentiated into roots. stems, and leaves, unicellular single-celled (yeast), composed of mycelium (filaments and masses of cells which make up the body, reproduce asexually and or sexually. Prepared by: RSCRUZ Centro Escolar University- Manila 14  Taxomomy- Classification Supplemental readings: Chapter 1 p 1-4 -BAILEY & SCOTT’S DIAGNOSTIC MICROBIOLOGY. 12 Ed. Forbes,BA, Sahm, DF, Weissfeld, AS. Six kingdom Classification Prepared by: RSCRUZ Centro Escolar University- Manila  Whittaker Five Kingdom Higher form of Organism Unicellular organism, eukaryote Unicellular, prokaryote Supplemental readings: Chapter 1 p 1-4 -BAILEY & SCOTT’S DIAGNOSTIC MICROBIOLOGY. 12 Ed. Forbes,BA, Sahm, DF, Weissfeld, AS. Prepared by: RSCRUZ Centro Escolar University- Manila  The Taxonomic Hierarchy : Define Species Genus Family Order Class Phylum Kingdom Domain Prepared by: RSCRUZ Centro Escolar University- Manila 17  Some the genus name is abbreviated by using the first letter of the genus followed by a period and the species of epithet. e.g., S. aureus The abbreviation “sp.” is used to designate single species. e.g., Penicillium sp. The abbreviation “spp.” is used to designate more than one species. e.g., Staphylococcus spp. Nicknames and slang terms frequently used with in hospitals are GC and gonococci (for Neisseria gonorrhoeae), meningococci (for Neisseria meningitidis). Prepared by: RSCRUZ Centro Escolar University- Manila 18 Prepared by: RSCRUZ Centro Escolar University- Manila 19  Species Name By convention: – first word of binomial name is genus and is always capitalized – second word refers to specific epithet and is not capitalized together form scientific name, written in italics – a complete scientific name may includes the author’s name. Ex. Bacillus anthracis 60 MINS Prepared by: RSCRUZ Centro Escolar University- Manila 20  Understanding the Concept: Describe the 5 Groups of microorganisms. Give 5 examples for each group and the disease associated 4 Types of bacterial stains Type of bacteria based on Gram’s staining Types of Culture media based Used Prepared by: RSCRUZ Centro Escolar University- Manila 21 Prokaryotic and Eukaryotic Difference Prokaryotic Eukaryotic Cell Membrane Chromosome Absent Possess a “true nucleus” it unifies, controls, and integrates the functions of the entire cell ; command center of the cell Cytoplasm A semifluid cytoplasm of Consists of semifluid, prokaryotic cells consist gelatinous, nutrient of water, enzymes matrix. Within the dissolved oxygen, waste cytoplasm are found products, essential insoluble storage nutrients, proteins, granules and variety of carbohydrates, and lipids. cytoplasmic organelles. Prepared by: RSCRUZ Centro Escolar University- Manila 22 Prokaryotic and Eukaryotic Features Plant type Animal Type Prokaryotic Cell Biological All plants, fungi, and All animals and All bacteria distribution algae protozoa Nuclear membrane Present Present Absent Membranous Generally absent except structures and Present Present mesosomes and photosynthetic other than cell membrane membrane Present Present Microtubules Absent Cytoplasmic 80S 80S 70S Ribosomes When present, Flagella, when present, have When present, have a Flagella or cillia have a complex simple twisted protein structure; complex structure structure no cilia When present of simple Of complex chemical Cell Wall chemical constitution, Absent constitution, containing usually cellulose peptidoglycan Prepared by: RSCRUZ Centro Escolar University- Manila 23  Comparison of Human and Bacterial Classification Human Being Escherichia coli Staphylococcus aureus Kingdom (Domain) Animalia (Eucarya) Procaryotae (Bacteria) Procaryotae (Bacteria) Phylum Chordata Proteobacteria Firmicutes Class Mammalia Gammaproteobacteria Bacilli Order Primates Enterobacteriales Bacillales Family Homonidae Enterobacteriaceae Staphylococcaceae Genus Homo Escherichia Staphylococcus Species (a species has two Homo sapiens Escherichia coli Staphylococcus aureus name; the first name is the genus, and the second name is the specific epithet) Prepared by: RSCRUZ Centro Escolar University- Manila 24  Understanding the Concept: Differentiate the Prokaryotic and Eukaryotic Cell (Provide information/s on each category below.) Categories Prokaryotes Eukaryotes Group/s Genetic System Location Chromosome Nucleolus Sexuality Streaming Pinocytosis Mesosome Ribosome Mitochondria Chloroplast Golgi Apparatus E.reticulum Locomotor Cell wall Pseudopodia DNA base ratio mole % of G+C%) Supplemental readings: Chapter 2 p 21-25. BAILEY & SCOTT’S DIAGNOSTIC MICROBIOLOGY. 12 Ed. Forbes,BA, Sahm, DF, Weissfeld, Prepared by: RSCRUZ Centro Escolar University- Manila 25 Recap of Structural differences between Prokaryotic and Eukaryotic Cells Eukaryotic cells are divided into plant and animal types. Animal cells do not have cell wall, whereas plant cells have simple cell wall, usually containing cellulose. Cellulose, a type of polysaccharides, is a rigid polymer of glucose. Prokaryotic cells have complex cell walls consisting of proteins, lipids, and polysaccharide. Eukaryotic cells contain membranous structures and many membrane bound organelles. Prokaryotic cells possess no membrane other than the cell membrane that encloses the cytoplasm. Eukaryotic ribosome are larger and more dense than those found in prokaryotes Prepared by: RSCRUZ Centro Escolar University- Manila 26 Prokaryotic and Eukaryotic bacteria Prepared by: RSCRUZ Centro Escolar University- Manila 27 Label the parts of the bacterial cell (10 mins) Prepared by: RSCRUZ Centro Escolar University- Manila 28  Supplemental readings: Chapter 2 p 21-25. BAILEY & SCOTT’S DIAGNOSTIC MICROBIOLOGY. 12 Ed. Forbes,BA, Sahm, DF, Weissfeld, AS. Prepared by: RSCRUZ Centro Escolar University- Manila 29 Prepared by: RSCRUZ Centro Escolar University- Manila 30  Bacterial cell wall in relation to Gram Reaction Gram positive cell wall Gram negative cell wall Prepared by: RSCRUZ Centro Escolar University- Manila 31 Prepared by: RSCRUZ Centro Escolar University- Manila 32 Prepared by: RSCRUZ Centro Escolar University- Manila 33 Bacterial morphology Prepared by: RSCRUZ Centro Escolar University- Manila 34  Microbial Classification and Identification Criteria 2 general criteria Phenotypic - readily observable characteristics ( visually present) Genotypic - genetic makeup of the organisms (genes and nucleic acid manipulation) Prepared by: RSCRUZ Centro Escolar University- Manila 35  Microbial Classification and Identification Criteria Phenotypic Criteria 1. Macroscopic morphology / Cultural/Colonial) Characteristics 2. Microscopic morphology 3. Staining Characteristics 4. Environmental & Nutritional Requirement 5. Resistance profile/ Antimicrobial Testing 6. Antigenic profile/ Serological Testing 7. Subcellular or Biochemical properties/test Genotypic Criteria 1. DNA base composition Supplemental readings: Chapter 5 p 73-76. BAILEY & SCOTT’S 2. Nucleic Acid ( DNA and RNA) base sequence DIAGNOSTIC MICROBIOLOGY. 12 Ed. Forbes,BA, Sahm, DF, Weissfeld, Prepared by: RSCRUZ Centro Escolar University- Manila 36  Understanding the Concept: 1. How do you determine genotype and phenotype of the organism? (answer briefly) 2. State 3 specific examples of phenotypic and genotypic characteristics of a pathogen. Cite one bacterial species. 3. Create a phylogenetic tree of Escherichia coli, in relation to Homo sapiens, Balantidium coli and Candida albicans. Prepared by: RSCRUZ Centro Escolar University- Manila 37 Growth Curve ❑ During an initial LAG phase the rate of growth or cell division is very slow. ❑ Growth or cell division then starts to accelerate into the EXPONENTIAL phase. ❑ Growth will start to DECELERATE (DECLINE). ❑ This may be followed by a STATIONARY phase, during which there is no discernible change in cell concentration or biomass. ❑ Finally, we may observe a phase of CELL DEATH and LYSIS - which results in a decrease in cell number and/or biomass. Prepared by: RSCRUZ Centro Escolar University- Manila 38  Microscopy Brightfield Microscope. Magnification is obtained by a system of optical lenses. Compound Bright-field Microscope- equipped with two lenses and a visible light source that passes through the specimen and lenses to the observer's eye. The eyepiece contains the ocular lens, the next lens is in the objectives near the object to be viewed. Form a dark image against a brighter background (Resolving power - 0.2000 um.) Objectives: Low-power objectives- 100x, High-power objectives- 400X, Oil-immersion objectives -1,000X Resolving power -ability of the lens to distinguish two adjacent points or objects at a particular distance apart. It depends on the wavelenght of the light source and the numerical aperture. The numerical aperture is proportional to the resolving power. Supplemental readings: Chapter 6 p 78-92. BAILEY & SCOTT’S DIAGNOSTIC MICROBIOLOGY. 12 Ed. Forbes,BA, Sahm, DF, Weissfeld, Prepared by: RSCRUZ Centro Escolar University- Manila 39  Microscopy Dark-field Microscope- the light is directed toward the specimen from the side so that the only light to reach the objective is reflected from the bacteria or object to be studied. Thus, the microbes appear as bright object on a dark background. Prepared by: RSCRUZ Centro Escolar University- Manila 40  Microscopy Phase-Contrast Microscope. It is use to observe living microorganism without staining because the light refracted by the living cells is different from the surrounding medium to see the object more easily. Prepared by: RSCRUZ Centro Escolar University- Manila 41  Microscopy Fluorescence Microscope. This microscope uses ultraviolet ( UV) light source that illuminates the object without passing into the objective of the microscope. The ultraviolet light strikes certain dye (fluorescein isothiocyanate, rhodamine B ) and pegments they emit flourescent against a dark background. Prepared by: RSCRUZ Centro Escolar University- Manila 42  Microscopy Electron Microscope. It uses electron beam instead of visible light and magnets instead of lenses to focus the beam. The electron passes through the dry specimen, which is mounted in wax or plastic, and the image is seen on the fluorescent screen. a.Transmission electron microscope. The object can be viewed on the screen and allows examination of cellular ultrastructure, as well as viruses. Image is two dimensional. Resolving power- 0.0005 um. Maximum magnification - 200,000X Scanning electron microscope. It shows three dimensional view of specimen. it is also useful in examining surface structure of cells and viruses. Resolution is limited compared with transmission electron microscope. Resolving power- 0.0200 um. Maximum magnification- 10,000X. Prepared by: RSCRUZ Centro Escolar University- Manila 43  Electron Microscopy 20 mins Prepared by: RSCRUZ Centro Escolar University- Manila 44  Examples of Bacteria and Disease associated Disease Organs Affected Transmission Anthrax Blood, Lungs, Skin Soil Bacterial Conjunctivitis Eyes Contact Botulism Neuromuscular junction Food, Water Brucellosis Spleen, Lymph glands Food, Water Urethra, Cervix, Fallopian Tubes, Chlamydial urethritis Sexual Epididymis, Eyes, Pharynx Clostridial Food Poisoning Intestine Food, Water Diphtheria Blood, Skin, Upper respiratory tract, Air Heart, Nerve fibers Endemic Typhus (Murine Typhus) Blood, Skin Flea Epidemic Typhus (Typhus Fever) Blood, Skin Flea Flavobacterium meningitis Upper respiratory tract, Meninges Air Gas Gangrene Muscles, Nerves, Blood cells Soil Gonorrhea Urethra, Cervix, Fallopian Tubes, Sexual Epididymis, Eyes, Pharynx Haemophilus meningitis Upper respiratory tract, Meninges Air Klebsiella pneumonia Lungs Air Leprosy (Hansen's Disease) Epididymis skin, Bones, Peripheral Contact nerves Lymphogranuloma venereum Inguinal lymph nodes, Rectum Sexual Prepared by: RSCRUZ Centro Escolar University- Manila 45  Examples of Bacteria and Disease associated Meningococcal Meningitis Upper respiratory tract, Blood, Meninges Air Mycoplasmal urethritis Urethra, Fallopian tubes Sexual Pneumococcal pneumonia Lungs Air Primary Atypical Pneumonia Lungs Air Psittacosis Lungs Air Q Fever Lungs Air Relapsing Fever Blood, Liver Louse Rickettsialpox Blood, Skin Mite Rocky Mountain Spotted Fever Blood, Skin Tick Salmonellosis Intestine Food, Water Scrub Typhus Blood, Skin Mite Serratia pneumonia Lungs Air Shigellosis Intestine Food, Water Staphylococcal Food Poisoning Intestine Food, Water Staphylococcal skin diseases Skin Contact Syphilis Skin, Cardiovascular Organs Sexual Prepared by: RSCRUZ Centro Escolar University- Manila 46  Examples of Bacteria and Disease associated Tetanus Nerves at synapse Soil Tickborne Fevers Blood, Skin Tick Trachoma Eyes Contact Ureaplasmal urethritis Urethra,Fallopian tubes,Epididymis Sexual Vaginitis Vagina Sexual Prepared by: RSCRUZ Centro Escolar University- Manila 47 Bacterial Morphology Bacteria vary in size from 0.4 to 2μm. They occur in the three basic shapes. ❖ Cocci (spherical) ❖ Bacilli (rod-shaped) ❖ Spirochetes (spiral) Cocci may be seen singly or in pairs (diploccoci), chains (streptococci), clusters (staphylococci), packets of four (tetrads). Or packets of eight (octads), depending on the particular species and the manner in which cells divide. e.g Enterococcus spp., Neisseria spp., Staphylococcus spp., and Streptococcus spp. Prepared by: RSCRUZ Centro Escolar University- Manila 48  Bacilli may be short or long, thick or thin, pointed or with curved or blunts ends. From very short coccibacilli to long filamentous rods. Bacilli with tapered, pointed ends are termed fusiform. e.g include the members of the family Enterobacteriaceae (e.g Enterobacter, Escherichia, Klebsiella , Proteus, Salmonella, and Shigella spp.), Haemophilus influenzae, Pseudomonas aeruginosa, Bacillus spp., and Clostridium spp. Curved and spiral-shaped bacilli are placed into a third morphological grouping. e.g Vibrio spp., such as Vibrio cholerae and Vibrio parahaemolysticus Prepared by: RSCRUZ Centro Escolar University- Manila 49  Some bacteria may lose their characteristics shape because adverse growth conditions prevent the production of normal cells. Such as cell wall deficient bacteria are called L-forms. Some L-forms revert to their original shape when placed in favorable growth conditions, whereas others do not. Mycoplasma do not have cell walls; thus , microscopically they appear in various shape. The ability to exist in a variety of shapes is known as pleomorphism. Prepared by: RSCRUZ Centro Escolar University- Manila 50 ❖ Dr. Hans Christian Gram - developed a staining technique that bears his name. Gram Stain most important staining procedure in the bacteriology laboratory differentiates between “Gram-positive” and “Gram-negative” bacteria. Procedure: Crystal Violet (V) - primary stain (1-2 mins.) Gram’s Iodine (I) - mordant (1 min.) Alcohol (A) - until no blue color comes -off Safranin (S) - counterstain (30 sec.-1min) Supplemental readings: Chapter 6 p -82-83. BAILEY & SCOTT’S DIAGNOSTIC MICROBIOLOGY. 12 Ed. Forbes,BA, Sahm, DF, Weissfeld, Prepared by: RSCRUZ Centro Escolar University- Manila 51  Gram Staining procedure: Add the Crystal Violet dye to one edge of the coverslip “tunnel.” Draw the dye through using an absorbant paper towel applied to the opposite edge of the cover slip. Draw the dye through until all the water has been replaced by dye. Stain for 30 seconds. In a similar manner, wash the slide by drawing water under the coverslip with a paper towel until no further purple color is removed. Add the Gram's Iodine solution by adding several drops of the reagent to the same edge of the coverslip and drawing it through with a paper towel. Treat for about 1.5 minutes. Decolorize the slide by drawing 95% alcohol under the coverslip until no further purple due is removed. Wash the slide as in step b. Counter stain the slide by placing Safranin dye at the same end of the “tunnel” and drawing it through with a paper towel. Stain for 1/2 minute. Wash the slide as in step b. Observe the slide under high dry or oil immersion microscopy. Prepared by: RSCRUZ Centro Escolar University- Manila 52  Gram Reactions o Gram positive. If the bacteria ( stained with crystal violet) were not decolorized during the decolorization step appearing as blue-to- purple bacteria. o Thick layer of peptidoglycan makes it difficult to remove the crystal violet iodine complex during the decolorization step. o Gram-negative. The crystal violet was removed from the cells during the decolorization step, and the cells were subsequently stained by the safranin they will be pink-to-red. Thin layer of peptidoglycan in the cell walls of Gram-negative bacteria make it easier to remove the crystal violet. Some stains of bacteria are neither consistently blue-to-purple nor pink-to-red; they are referred to as Gram-variable bacteria. Prepared by: RSCRUZ Centro Escolar University- Manila 53  Acid Fast Staining Acid Fast Stain Carbol fuschin (bright red dye). Heat for 5mins. Acid Alcohol- until red color comes-off Methylene blue General Rule: Al bacteria are none or not acid fast except Mycobacterium, Nocardia- slightly acid fast. Prepared by: RSCRUZ Centro Escolar University- Manila 54 ACID FAST STAIN Contains wax-like lipoidal material affecting staining quality. Carbolfuchsin is primary stain. Acid fast organisms resist decolorization with acid alcohol. After decolorization, methyelene blue is added to organisms to counterstain any material that is not acid fast Prepared by: RSCRUZ Centro Escolar University- Manila 55 Types of Bacterial Staining Procedures Category Example(s) Purpose Simple staining Staining with Merely to stain cells so that their size, shape, and procedure methylene blue morphological arrangement can be determined Structural Capsule stains To determine if the organism is encapsuled staining procedures Flagella stain To determined if the organism posses flagella and, if so their number and location on the cell Endospore stains To determined if the organism is a spore-former and, if so, to determined if the spore are terminal or subterminal Differential Gram stain To differentiate between Gram-positive and staining Gram-negative procedures Acid-fast stain To differentiate between acid-fast and non-acid fast bacteria Prepared by: RSCRUZ Centro Escolar University- Manila 56  Acridine Orange It is a flourochrome dye that stains both Gram positive and Gram negative bacteria, living or dead. Used to locate bacteria in blood cultures and other specimens. Inhibition of Icmt induces autophagy in human prostate cancer cells. PC3 cell were treated with the small molecule inhibitor of Icmt, cysmethynil, for 48 h and then stained by acridine orange Prepared by: RSCRUZ Centro Escolar University- Manila 57  Calcofluor White It is fluorochrome that binds to chitin in fungal call walls. It fluoresces as a bright apple-green or blue-white, allowing visualization of fungal structures with fluorescent microscope. A pseudohyphal mutant of the yeast Cryptococcus neoformans. Cell walls are stained blue with calcofluor white, and nuclei are stained with sytox green. Prepared by: RSCRUZ Centro Escolar University- Manila 58 Methylene Blue Traditionally used to stain Corynebacterium diphtheriae for observation of metachromatic granules. It is also used as counterstain in the acid-fast staining procedures Lambic Prepared by: RSCRUZ Centro Escolar University- Manila 59  Lactophenol Cotton Blue Used to stain the cell walls of medically important fungi grown in slide culture. Prepared by: RSCRUZ Centro Escolar University- Manila 60 Endospore Stain Malachite green, the primary stain is applied and heated to steaming for about 5 min. Washed for 30 sec. to remove the primary stain Applied the counterstain safranin to the smear It appears green, within pink-or-appearing bacterial cells Prepared by: RSCRUZ Centro Escolar University- Manila 61  India Ink It is a negative stain used to visualize capsules surrounding certain yeasts, such as Cryptococcus spp. The fine particles are excluded from the capsule. Leaving a dark background and a clear capsule surrounding the yeast. Bacterial capsules outlined by India ink viewed by light microscopy. This is a true capsule, a discrete layer of polysaccharide surrounding the cells. Prepared by: RSCRUZ Centro Escolar University- Manila 62 Microbial Growth and Nutrition 3 major nutritional needs for growth ❖ source of carbon (for making cellular constituents). 50% of the dry weight of a bacterium ❖ source of nitrogen (for protein synthesis) 14% of the dry weight ❖ source of energy (ATP, for performing cellular functions). 4% are : Phosphate for nucleic acids and phospholipids of cell membranes, and sulfur for protein synthesis Prepared by: RSCRUZ Centro Escolar University- Manila 63  Colonial Characteristics Bacterial Colony contains millions of organism. determine the size, color, overall shape, elevation, and appearance of the edge or margin of the colony. also the results of enzymatic activity on various types of culture media. changes in the color of the medium or colonial features serve as “clues” in identification of bacteria. Supplemental readings: Chapter 7 p 93-119. BAILEY & SCOTT’S DIAGNOSTIC MICROBIOLOGY. 12 Ed. Forbes,BA, Sahm, DF, Weissfeld, Prepared by: RSCRUZ Centro Escolar University- Manila 64 Nutritional requirements for growth Nutritional requirement a. Autotrophic or lithotrophic- carbon dioxide as the sole source of carbon, with only H2O and inorganic salts required. b. Heterotrophic or organotrophic- requires more complex substances for growth. -require an organic source of carbon. Types of Medium based on Microbial Requirement Minimal medium- laboratory growth medium whose contents are simple. Nutrient media- more complex and made of extracts of meat or soybeans Selective media- media containing additives that inhibit the growth of some bacteria but allows others to grow. Prepared by: RSCRUZ Centro Escolar University- Manila 65 Differential media- ingredients that allow visualization of metabolic differences between groups or species of bacteria. Transport medium- when a delay between collection of the specimen and culturing the specimen is necessary. Prepared by: RSCRUZ Centro Escolar University- Manila 66  Atmospheric /Oxygen Requirement Carbon Oxygen dioxide MAJOR GROUPS Obligate Facultative Microaerop Aerotolerant Obligate Capnophilic aerobes aerobes hilic aerobes anaerobes Require either with oxygen survive does not require best grow when Oxygen or without for in the presence oxygen the atmospheric oxygen multiplication of oxygen is enriched Prepared by: RSCRUZ Centro Escolar University- Manila 67  Bacterial Growth ❑ Generation Time - Bacteria replicate by binary fission -Generation time or doubling time time required for one cell to divide into cells -20 minutes for the bacteria in the culture to generate for fats- growing organism e.g., E. coli -24 hours for a slowing bacterium e.g., Mycobacterium tuberculosis Prepared by: RSCRUZ Centro Escolar University- Manila 68 Bacterial Biochemistry and Metabolism Metabolism - breakdown organic compounds to synthesize new bacterial parts from the resulting carbon skeleton Fermentation and Respiration obligate and facultative anaerobes less efficient in energy generation than respiration 2 important diagnostic test used in identification of the Enterobacteriaceae Voges-Proskauer (VP) Methyl red tests molecular oxygen is the final receptor Certain anaerobes can carry out anaerobic respiration such as Nitrate Sulfate final electron receptor Prepared by: RSCRUZ Centro Escolar University- Manila 69  Some bacteria use O2 in the air to oxidize inorganic compounds and produce ATP (energy). The energy is enough to convert CO2 into organic material needed for cell growth Thiobacillus (sulfur S) Nitorsomonas (ammonia) Nitrobacter (nitrite) – Various genera (hydrogen etc.) Prepared by: RSCRUZ Centro Escolar University- Manila 70  –Final electron acceptor : never be O2 ▪Sulfate reducer: final electron acceptor is sodium sulfate (Na2 SO4) ▪Methane reducer: final electron acceptor is CO2 ▪Nitrate reducer : final electron acceptor is sodium nitrate (NaNO3) O2/H2O coupling is the most oxidizing, more energy in aerobic respiration. Therefore, anaerobic is less energy efficient. Prepared by: RSCRUZ Centro Escolar University- Manila 71 Prepared by: RSCRUZ Centro Escolar University- Manila 72 Summary of Supplemental Lecture (SL) Part 1: 1. 1st Virtual Orientation 11 Aug 2020 2. Introduction to Clinical Bacteriology 3. Taxonomy and Classification 4. Prokaryotic and Eukaryotic Cells 5 Ways of Studying Microorganism a. Morphology ( forms and shapes), arrangement, Staining reaction b. Microscopy c. Cultural/ Colonial and Environmental/ Physiological Characteristics It seems that my Supplemental lectures run so long, I will continue my lecture on Part 2, covering the following topics: Part 2. 3. Biochemical / Enzymatic Test 4. Antimicrobial Susceptibility Test Prepared by: RSCRUZ Centro Escolar University- Manila 73