General Microbiology Lecture Notes PDF

General Microbiology (Lecture) rosmo Microorganisms Bacterial Cell Structure...

General Microbiology (Lecture) rosmo Microorganisms Bacterial Cell Structure Domains of Cellular Microorganisms Gram Positive vs. Gram Negative eukaryotes prokaryotes gram positive gram negative nucleus present no true nucleus thickness 20-25nm 11-15nm periplasmic space peptidoglycan/murein 50%-90% ATP production site mitochondria in cell membrane – flexibility of cell wall connected by 10% no. of ATP/glucose 36 38 – sugar and protein cross-bridging 80S 70S lipopolysaccharide absent present ribosome 60S LSU ; 40S SSU 50S LSU ; 30S SSU present between double-stranded double stranded periplasmic space absent outer CM-murein- DNA structure linear circular inner CM DNA histone teichoic acid present absent present (octamer) none – binds/coils DNA no. of rings in basal two four DNA location nucleus cytoplasm/nucleoid body of flagella – fungi bacteria and lipid content low high (11-22%) multicellular molds, archaebacteria protoplast (PDG is spheroplast (cannot action of lysozyme unicellular yeasts easily destroyed) penetrate LPS layer) domains – protozoa endotoxin unicellular parasite, – component of lipid commensals A in outer CM of LPS toxins produced exotoxin – cause inflammation – algae (redness, pain, fever) Acellular Microbes CFS  meningitis effects in sterile fluid none blood  sepsis virus prions examples ▪ Bacillus ▪ Pseudomonas replicates only living cells proteins that trigger normal ▪ Clostridium aeruginosa protein to fold abnormally ▪ Listeria ▪ Klebsiella – non-cellular infectious agents monocytogenes pneumoniae – no own metabolic machinery ▪ Staphylococcus ▪ Vibrio cholerae – intracellular parasites that seek host ▪ Streptococcus ▪ Neisseria gonorrhoeae Polymerase Chain Reaction Structures Always Present in Microorganisms DNA polymerase – enzyme for industrial application a. cell wall d. cytoplasm – amplifies specific DNA segments b. cell surface membrane e. ribosomes – produced by Pyrococcus furiosus and Thermus aquaticus, which are archaea thermophiles found in hot springs c. chromosomal DNA – vital, dictates cellular functions – polymer: complex chemical source of ligase in mol. engr. Peptidoglycan with subunits of polysaccharide, – composed of N-acetyl muramic acid and N-acetyl glucosamine polypeptide, lipid, and molecular glue that joins polynucleotide target and vector DNA – has waxy mycolic/hydroxymethoxy acid that resists staining – complex chemical with biomolecules as subunits major component of Mycobacterium tuberculosis cell wall, RT-PCR – test for COVID-19, which is caused by SARS-COV2 (RNA virus) in which, acid fast staining by Ziehl Neelsen is conducted – ACE-2 receptors: angiotensin-converting enzyme 2 Flagella : receptor in epithelial tissue of nose and mouth where virus binds atrichous – no flagella; immotile lopotrichous – on one pole Note: Virus has only one type of nucleotide (DNA or RNA). monotrichous – one flagellum amphitrichous – both poles Restriction Enzymes peritrichous – many flagella restriction enzymes – molecular scissors Plasmids – can produce recombinant DNA for hepatitis B and insulin – double-stranded, circular, covalently-bound extrachromosomal DNA – bacteria involved – common in gram (-); plasmid genes have no vital function a. Hind III (Haemophilus influenzae) F plasmid – codes for pili b. Bam HI (Bacillus amyloliquifaciens) R plasmid – codes for antibacterial/antimicrobial resistance genes c. Hae III (Haemophilus aegyptius) Note: Antibiotic residue-free poultry prevents antibiotic resistance, d. Alu I (Arthrobacter luteus) for overused antibiotics increases bacterial resistance. e. EcoRi (Escherichia coli) Clavulanic acid stops bacteria from breaking down antibiotics. Note: Enzyme’s denature at 56o-60OC, as it coagulates. Nosocomial infection is a hospital-acquired infection of P. aeruginosa, Application of Bacteria in the Food Industry a biofilm (opportunistic group of bacteria attached to surfaces). lactic acid bacteria – fermentation/preservation of food Pili ▪ Corynebacterium glutanicum – source of MSG – extracellular appendages for attachment to other cells ▪ Corynebacterium diphteriae – harmful and translocation of DNA alcohol dehydrogenase – tolerance to alcohol; women have less – bacterial conjugation: alc. deh. transferring of gene via pili – C2H3OH  acetaldehyde  acetic acid F+ bacteria with pilus Beta-Lactams – F for fertility, donor – major groups F- bacteria without pilus – recipient a. Penicillins c. Monobactam Endospores b. Cephalosporins d. Carbapenems – has calcium dipicolinate antibiotics – destroys peptidoglycan of Beta-lactam resistant structure in Bacillus and Clostridium lysozyme – found in saliva and tears for digesting peptidoglycan – vegetative cell becomes spore-forming when: Methicillin-resistant Staphylococcus aureus bacteria (MiRSA) a. deprived of nutrients c. starvation – beta-lactam that is resistant to antibiotics b. aging of culture d. desiccation 1 out of 4 General Microbiology (Lecture) rosmo Capsule Physiological Characterizations – additional protection that delays phagocytosis of monocytes 1. energy source – phototroph, chemotroph engulf can’t digest 2. carbon source – heterotrophs: uses sugar for C – capsule  monocyte  lysosome  macrophages – lithotrophs: uses inorganic C for electron and energy – virulence factor – e.g. ▪ Streptoccocus pneumoniae – deadly lobar pneumonia : e.g. iron bacteria in red, ferrous soil degree of pathogenicity ▪ Mycoplasma pneumoniae 3. temperature – walking/atypical pneumonia Molecular Characterizations ability to cause disease ▪ Neisseria meningitidis 1. rRNA gene sequencing – 16S bacteria, 18S unicellular eukaryotes – sialic acid capsule smooth, encapsulated 2. Svedberg unit – sedimentation constant ▪ Streptococcus pyogenes – hyaluronic acid capsule 3. G:C ratio – guanine to cytosine Other Parts Colonial Characterization infolding of cell surface membrane – for photosynthesis and N-fixation 1. colony size – pinpoint, pinhead, small, medium, large encapsulated bacteria – virulent 2. colony color – buff, yellow, white, green – e.g. Streptococcus pneumoniae – rough form/strain – Pseudomonas aeruginosa: blue green, water soluble pigment (pyoverdin, pyocyanin) that diffuses with agar Primary Metabolites vs. Secondary Metabolites : cause of green phlegm and Otitis media (ear inflammation) primary secondary metabolic growth, defense mechanism – Serratia marcescens: red, water insoluble pigment (prodigiosin) importance reproduction 3. colony elevation – flat, raised, concave, convex produced by metabolism primary metabolites Biochemical Tests for Gram Positive Cocci logarithmic stationary phase of growth (active division) (due to lack of nutrients 1. catalase test – differentiating Staphylococci from Streptococci and environmental stress) – positive: bubble formation H2O2  catalase  H20 + O2 alcohol, antibiotics Staphylococci are catalase positive. amino acid, (e.g. Bacteriocin, example glutamic acid Penicillin 2. coagulase test – pathogenic and non-pathogenic Staphylococci discovered by Fleming – requires fresh rabid plasma reagent from Penicillium notatum) – positive: Staphylococcus aureus: acne, pimple, folliculitis, Bacterial Reproduction carbuncle, furuncle, boils, and food poisoning – negative: Staphylococcus epidermidis: normal skin flora Stages in Bacterial Growth that may cause stitch abscess Note: Bacteria reproduce asexually via binary fission, Biochemical Tests for Gram Negative Bacilli in which cells divide over a certain time. (Family Enterobacteriaceae) 1. lag phase – acclimatization/period of adjustment 1. indole test – detects tryptophanase of coliform – new environment = culture media – tryptophan ---hydrolyzed pyruvic acid, ammonia, indole 2-4. log phase  stationary/plateau phase  death/decline phase – reagent: Incubation a. Ehrlich’s reagent: 0.5 mL (paradimethylaminobenzaldehyde) Note: Optimal condition for growing bacteria in incubator is : for anaerobes and weak indole producers 35-37OC for 18-24 hours OR 24OC for more than 3 days b. Kovac’s reagent: for aerobes turbidity of medium = positive results – pH indicator: red Non-fastidious bacteria generate within 20 minutes, while slow growers, such as Tubercle bacilli, appear more than 20 hours. 2. methyl red test – detects ability to perform mixed acid fermentation – broth: MRVP glucose phosphate broth Methods of Studying Microorganisms in the Laboratory – pH indicator: (methyl red  acidic ; yellow  alkaline) Key Notes 3. Voges Prauskauer test – detects bacteria-producing Acetoin ▪ substrate – reagent/substance acted upon by enzyme (acetylmethyl carbinol) from glucose fermentation strong alkali ▪ antiseptic – chemical applied on living tissue for sterilizing – pH indicator: Acetoin + napthol  diacetyl + guanidine ▪ disinfectant – chemical applied on inert surface (e.g. Chlorox)  methyl red (positive) or beige yellow (negative) ▪ colony forming unit – many cells of same species – broth: MRVP broth – for counting bacteria: CFU/mL – reagent: ▪ plasma – liquid when anticoagulant is added to prevent clotting a. Barritt’s A: α-napthol for color intensifier ▪ serum – liquid that remains after clotting of blood b. Barritt’s B: 40% KOH ▪ All cocci are gram positive, except Neisseria, Veilonella, Moraxella. 4. citrate test – detects citrate permease ▪ All bacilli are gram negative, except Bacillus, Listeria, – detects sodium citrate as C source Clostridium, Corynebacterium, etc. – detects ammonium dihydrogen phosphate as N source ▪ RBCs have numerous catalase. – citrate  pyruvate ▪ Skin has many Staphylococcus. – agar: Simmon Citrate agar slant ▪ Multiple interrupted streaking isolate single colonies – pH indicator: Bromthymol blue, >7.6 pH Phenotypic Characterizations : e.g. Salmonella, Providencia, Edwardsiella, 1. gram staining reactions – purple (+) and red (-) Enterobacter, Citrobacter, Klebsiella, Serratia, 2. bacterial shapes – negative: slant remains green 3. cell arrangement : e.g. Proteus, Escherichia, Yersinia, 4. motility – monotrichous (e.g. Vibrio cholerae) Morganella, Shigella – peritrichous (e.g. Escherichia coli) 5. triple sucrose iron agar (slant butt agar) – atrichous (e.g. Shigella dysenteriae – bacillary dysentery) – differentiates enterics that ferment CHO and reduce S 2 out of 4 General Microbiology (Lecture) rosmo – requires 3 CHO (glucose, lactose, sucrose), Fe ions, Gram Staining Technique sodium thiosulfate, and phenol red for pH indicator 1. crystal violet – initial staining; targets cell wall’s peptidoglycan – yellow (A/A): 2 or more sugars fermented, acid as by-product 2. gram’s iodine – mordant : e.g. a. lactose fermenters (increases affinity of dye to cell; color enhancer/intensifier) a.1. Escherichia coli 3. ethyl alcohol – decolorizer a.2. Klebsiella pneumoniae – encapsulated, mucoidal 4. safranine – counter staining, produces red a.3. Enterobacter aerogenes positive = blue negative = red – red slant-yellow butt (K/A): one sugar (glucose) fermenter Acid Fast Staining Technique by Ziehl Neelsen a. Serratia marcescens – detects Mycobacterium b. Salmonella, Shigella, Proteus, Providencia, Acinetobacter – requires sputum to diagnose pulmonary tuberculosis – red (K/K): non-sugar fermenter 1. carbol fuchsin – initial staining a. Pseudomonas 2. phenol – mordant that is already added to carbol fuchsin – (G+): due to displacement of bubble 3. acid alcohol – decolorizer – blackening (H2S+): producing hydrogen sulfide 4. malachite green/methylene blue – counter staining Types of Culture Medium positive AFP = red negative NAFB = blue/green primary isolation – simple (e.g. NA/NB) Describing a Bacteria enriched/enrichment – isolation of fastidious bacteria GS reaction  cell shape  cell arrangement  spores – e.g. BHIA/BHIB 1. positive 1. cocci 1. diplo (pair) 1. central differential – differentiate colonies (LF/NLF) and hemolytic pattern 2. negative 2. bacilli 2. tetrad 2. terminal – e.g. a. McConkey agar – growth medium for gram (-) 3. spirilla 3. cluster 3. subterminal 4. sarcina (8) – inhibitors of gram (+): bile salts, crystal violet 5. filament – pH indicator: neutral red 6. singly – positive: red, acidic, LF Physiological Needs of Bacteria – negative: colorless, alkaline, NLF Oxygen b. eosin methylene blue – detects E. coli – positive: greenish metallic, fish-eye colonies c. blood agar plate – hemolytic patterns ▪ alpha – Strep. pneumoniae Note: In humans, too much O causes blindness. ▪ beta – suspected Staph. aureus, Strep. agalactiae, obligate aerobes – has catalase and peroxidase that catabolize O Strep. pyogenes, List. ivanovii by degrading toxic reactive O species (ROS or free radicals) metabolites ▪ gamma – no hemolysis; LF – if ROS accumulates, intoxication occurs due to toxic O metabolites special – transportation of bacteria – use cotton plug for closure – e.g. a. potato/Sabouraud dextrose agar – fungal isolation – e.g. 1. all Bacillus b. thiosulfate-citrate-bile salt-sucrose agar – for Vibrio spp. a. B. subtilis – common lab contaminant Dyes – antibiotic Bacitracin Bacteriocin (enteric bacteria killer) Types of Dyes b. B. anthracis – largest pathogenic bacteria positive – stains the cell/tissue – cutaneous Anthrax: due to sheep raising – attracted to negative sialic acid on cell surface : skin penetration causes black Eschar – e.g. methylene blue, malachite green, crystal violet, safranine – respiratory Anthrax: bioweapon – types 1. simple – 1 dye – gastrointestinal Anthrax: deadliest; diarrhea 2. differential – 2 or more dyes c. B. cereus – fried rice poisoning – gram staining techniques d. B. thuringiensis – anti-pests – hematoxylin and eosin (histology) 2. Mycobacterium tuberculosis – chronic infection negative – stains background due to repulsion, thus cells are colorless – early morning sputum for sample – e.g. India ink, nigrossin – 3 successive negative results / days for clearance – uses 1. detects fungal meningitis from Cryptococcus neoformans – causes a. low-grade fever esp. afternoon fever, headache, stiff neck b. loss of appetite tracked through mouth and nose c. sudden weight loss transmitted through air d. cough for more than 3 weeks air dry to observe, NOT heat fix 3. Streptococcus pneumoniae stained bacterial smear to be viewed in OIO obligate anaerobes – NO catalase and peroxidase 2. detects bacterial meningitis from Haemophilus influenzae – sediments growing in broth culture major cause of meningitis in newborn, which is determined by hazy CSF – use cork for closure and treated via lumbar tap – e.g. 1. C. perfringens – gangrenous legs of diabetic patients 3. detects Bacilli (e.g. coliform) which are treated by hyperbaric oxygen – type 1. special stain 2. C. tetani – tennis racket bacilli that cause tetanus/lockjaw a. infectious particles 3. C. botulinum – canned good bacilli that cause botox 4. C. difficile – cause of pseudomembranous colitis, 3 out of 4 General Microbiology (Lecture) rosmo which is treated by Clindamycin facultative anaerobes – aerobes adapted in few O-environments – e.g. 1. coliform/enteric/colon bacteria a. Escherichia coli – greenish metallic on agar plate b. Salmonella typhi c. Enterobacter aerogenes microaerophiles – can live in small amount of O – e.g. 1. Helicobacter pylori 2. Campylobacter jejuni Temperature Note: Optimal condition for growing bacteria in incubator is 35-37OC for 18-24 hours OR 24OC for more than 3 days. mesophiles – 30-40oC room temperature bacteria – e.g. 1. Salmonella typhi – pathogenic psychrophiles – 10-20oC cold-loving bacteria – refrigerators slow down bacteria reproduction – e.g. 1. Listeria monocytogenes – multiples in refrigerators thermophiles – 100-600oC – pyrogens: bacteria that withstand very high temperature – e.g. 1. Pyrococcus furiosus 2. Thermus aquaticus pH acidophiles – e.g. 1. Lactobacillus casei strain Shirota 2. Cariogenic bacteria – tooth decay or dental caries 3. fungi (Candida albicans – vaginal and oral thrush) alkalinophiles – e.g. 1. Vibrio spp. – uses TCBS media (Thiosulfate–citrate–bile salts–sucrose agar with 8.6 pH) a. Vibrio parahemolyticus – shellfish food poisoning b. Vibrio cholerae – rice watery stool cholera CO2 Requirement capnophiles – bacteria that need increased CO2 tension – e.g. 1. Streptoccocus pneumoniae – fastidious bacteria that causes deadly lobar pneumonia 2. Helicobacter pylori – ulcer 3. Campylobacter jejuni – gastroenteritis osmophiles – can tolerate high osmotic medium – plasmolysis: hypertonic  cell shrinks – plasmoptysis: hypotonic  cell swells halophiles – bacteria that prefer high salt medium (10% NaCl) Honey Kills Bacteria! Due to honey’s high production of enzymatic hydrogen peroxide and high sugar content, cells are hypertonic and will undergo plasmolysis. 4 out of 4