General Microbiology (Lecture)

General Microbiology (Lecture) Microorganisms Bacterial Cell Structure Domains of Cellular Microorganisms Gram Positive vs. Gram Negative eukaryotes present ATP production site mitochondria no. of ATP/glucose 36 80S 60S LSU ; 40S SSU double-stranded linear prokaryotes no true nucleus periplasmic space in cell membrane 38 70S 50S LSU ; 30S SSU double stranded circular present (octamer) none nucleus cytoplasm/nucleoid bacteria and archaebacteria nucleus ribosome DNA structure DNA histone – binds/coils DNA DNA location – fungi multicellular molds, unicellular yeasts domains – protozoa unicellular parasite, commensals gram positive 20-25nm 50%-90% connected by cross-bridging absent thickness peptidoglycan/murein – flexibility of cell wall – sugar and protein lipopolysaccharide periplasmic space absent teichoic acid no. of rings in basal body of flagella lipid content present two four low protoplast (PDG is easily destroyed) high (11-22%) spheroplast (cannot penetrate LPS layer) endotoxin – component of lipid A in outer CM of LPS – cause inflammation (redness, pain, fever) CFS  meningitis blood  sepsis ▪ Pseudomonas aeruginosa ▪ Klebsiella pneumoniae ▪ Vibrio cholerae ▪ Neisseria gonorrhoeae action of lysozyme toxins produced exotoxin effects in sterile fluid virus replicates only living cells prions proteins that trigger normal protein to fold abnormally – non-cellular infectious agents – no own metabolic machinery – intracellular parasites that seek host Polymerase Chain Reaction DNA polymerase – enzyme for industrial application – amplifies specific DNA segments – produced by Pyrococcus furiosus and Thermus aquaticus, which are archaea thermophiles found in hot springs – polymer: complex chemical with subunits of polysaccharide, polypeptide, lipid, and polynucleotide examples none ▪ Bacillus ▪ Clostridium ▪ Listeria monocytogenes ▪ Staphylococcus ▪ Streptococcus Structures Always Present in Microorganisms a. cell wall d. cytoplasm b. cell surface membrane e. ribosomes c. chromosomal DNA – vital, dictates cellular functions Peptidoglycan source of ligase in mol. engr. molecular glue that joins target and vector DNA – complex chemical with biomolecules as subunits RT-PCR – test for COVID-19, which is caused by SARS-COV2 (RNA virus) – composed of N-acetyl muramic acid and N-acetyl glucosamine – has waxy mycolic/hydroxymethoxy acid that resists staining major component of Mycobacterium tuberculosis cell wall, in which, acid fast staining by Ziehl Neelsen is conducted – ACE-2 receptors: angiotensin-converting enzyme 2 : receptor in epithelial tissue of nose and mouth where virus binds Note: Virus has only one type of nucleotide (DNA or RNA). 10% present present between outer CM-mureininner CM absent – algae Acellular Microbes gram negative 11-15nm Flagella atrichous – no flagella; immotile lopotrichous – on one pole monotrichous – one flagellum amphitrichous – both poles peritrichous – many flagella Restriction Enzymes 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, for overused antibiotics increases bacterial resistance. d. Alu I (Arthrobacter luteus) e. EcoRi (Escherichia coli) Note: Enzyme’s denature at Clavulanic acid stops bacteria from breaking down antibiotics. 56o-60OC, as it coagulates. Application of Bacteria in the Food Industry Nosocomial infection is a hospital-acquired infection of P. aeruginosa, a biofilm (opportunistic group of bacteria attached to surfaces). Pili lactic acid bacteria – fermentation/preservation of food ▪ Corynebacterium glutanicum – source of MSG ▪ Corynebacterium diphteriae – harmful alcohol dehydrogenase – tolerance to alcohol; women have less alc. deh. – C2H3OH  acetaldehyde  acetic acid Beta-Lactams b. Cephalosporins – bacterial conjugation: transferring of gene via pili F+ bacteria with pilus – F for fertility, donor F- bacteria without pilus – recipient – major groups a. Penicillins – extracellular appendages for attachment to other cells and translocation of DNA Endospores c. Monobactam d. Carbapenems antibiotics – destroys peptidoglycan of Beta-lactam lysozyme – found in saliva and tears for digesting peptidoglycan Methicillin-resistant Staphylococcus aureus bacteria (MiRSA) – beta-lactam that is resistant to antibiotics – has calcium dipicolinate resistant structure in Bacillus and Clostridium – vegetative cell becomes spore-forming when: a. deprived of nutrients c. starvation b. aging of culture d. desiccation 1 out of 4 General Microbiology (Lecture) Capsule Physiological Characterizations – additional protection that delays phagocytosis of monocytes engulf – capsule can’t digest monocyte  lysosome  – virulence factor  macrophages – e.g. ▪ Streptoccocus pneumoniae – deadly lobar pneumonia degree of pathogenicity ability to cause disease smooth, encapsulated 1. energy source – phototroph, chemotroph 2. carbon source – heterotrophs: uses sugar for C – lithotrophs: uses inorganic C for electron and energy : e.g. iron bacteria in red, ferrous soil ▪ Mycoplasma pneumoniae – walking/atypical pneumonia 3. temperature ▪ Neisseria meningitidis – sialic acid capsule 1. rRNA gene sequencing – 16S bacteria, 18S unicellular eukaryotes ▪ Streptococcus pyogenes – hyaluronic acid capsule 3. G:C ratio – guanine to cytosine Molecular Characterizations 2. Svedberg unit – sedimentation constant Colonial Characterization Other Parts 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 Primary Metabolites vs. Secondary Metabolites importance produced by phase of growth primary metabolic growth, reproduction metabolism logarithmic (active division) alcohol, amino acid, glutamic acid example secondary defense mechanism primary metabolites stationary (due to lack of nutrients and environmental stress) antibiotics (e.g. Bacteriocin, Penicillin discovered by Fleming from Penicillium notatum) Bacterial Reproduction Stages in Bacterial Growth – Pseudomonas aeruginosa: blue green, water soluble pigment (pyoverdin, pyocyanin) that diffuses with agar : cause of green phlegm and Otitis media (ear inflammation) – Serratia marcescens: red, water insoluble pigment (prodigiosin) 3. colony elevation – flat, raised, concave, convex Biochemical Tests for Gram Positive Cocci 1. catalase test – differentiating Staphylococci from Streptococci – positive: bubble formation 2. coagulase test – pathogenic and non-pathogenic Staphylococci – requires fresh rabid plasma reagent – positive: Staphylococcus aureus: acne, pimple, folliculitis, carbuncle, furuncle, boils, and food poisoning – negative: Staphylococcus epidermidis: normal skin flora that may cause stitch abscess Note: Bacteria reproduce asexually via binary fission, in which cells divide over a certain time. 1. lag phase – acclimatization/period of adjustment – new environment = culture media 2-4. log phase  stationary/plateau phase  death/decline phase Incubation Note: Optimal condition for growing bacteria in incubator is 35-37OC for 18-24 hours OR 24OC for more than 3 days turbidity of medium = positive results Non-fastidious bacteria generate within 20 minutes, while slow growers, such as Tubercle bacilli, appear more than 20 hours. Methods of Studying Microorganisms in the Laboratory Key Notes ▪ substrate – reagent/substance acted upon by enzyme ▪ antiseptic – chemical applied on living tissue for sterilizing H2O2  catalase  H20 + O2 Staphylococci are catalase positive. Biochemical Tests for Gram Negative Bacilli (Family Enterobacteriaceae) 1. indole test – detects tryptophanase of coliform – tryptophan ---hydrolyzed pyruvic acid, ammonia, indole – reagent: a. Ehrlich’s reagent: 0.5 mL (paradimethylaminobenzaldehyde) : for anaerobes and weak indole producers b. Kovac’s reagent: for aerobes – pH indicator: red 2. methyl red test – detects ability to perform mixed acid fermentation – broth: MRVP glucose phosphate broth – pH indicator: (methyl red  acidic ; yellow  alkaline) 3. Voges Prauskauer test – detects bacteria-producing Acetoin (acetylmethyl carbinol) from glucose fermentation strong alkali ▪ disinfectant – chemical applied on inert surface (e.g. Chlorox) – pH indicator: Acetoin + napthol  diacetyl + guanidine  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. ▪ All bacilli are gram negative, except Bacillus, Listeria, Clostridium, Corynebacterium, etc. ▪ RBCs have numerous catalase. ▪ Skin has many Staphylococcus. ▪ Multiple interrupted streaking isolate single colonies Phenotypic Characterizations 1. gram staining reactions – purple (+) and red (-) 2. bacterial shapes 3. cell arrangement 4. motility – monotrichous (e.g. Vibrio cholerae) – peritrichous (e.g. Escherichia coli) – atrichous (e.g. Shigella dysenteriae – bacillary dysentery) 4. citrate test – detects citrate permease – detects sodium citrate as C source – detects ammonium dihydrogen phosphate as N source – citrate  pyruvate – agar: Simmon Citrate agar slant – pH indicator: Bromthymol blue, >7.6 pH : e.g. Salmonella, Providencia, Edwardsiella, Enterobacter, Citrobacter, Klebsiella, Serratia, – negative: slant remains green : e.g. Proteus, Escherichia, Yersinia, Morganella, Shigella 5. triple sucrose iron agar (slant butt agar) – differentiates enterics that ferment CHO and reduce S 2 out of 4 General Microbiology (Lecture) – requires 3 CHO (glucose, lactose, sucrose), Fe ions, sodium thiosulfate, and phenol red for pH indicator – yellow (A/A): 2 or more sugars fermented, acid as by-product : e.g. a. lactose fermenters Gram Staining Technique 1. crystal violet – initial staining; targets cell wall’s peptidoglycan 2. gram’s iodine – mordant (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 positive = blue a.3. Enterobacter aerogenes Acid Fast Staining Technique – red slant-yellow butt (K/A): one sugar (glucose) fermenter a. Serratia marcescens b. Salmonella, Shigella, Proteus, Providencia, Acinetobacter – red (K/K): non-sugar fermenter a. Pseudomonas – (G+): due to displacement of bubble – blackening (H2S+): producing hydrogen sulfide Types of Culture Medium by Ziehl Neelsen – detects Mycobacterium – requires sputum to diagnose pulmonary tuberculosis 1. carbol fuchsin – initial staining 2. phenol – mordant that is already added to carbol fuchsin 3. acid alcohol – decolorizer 4. malachite green/methylene blue – counter staining positive AFP = red primary isolation – simple (e.g. NA/NB) enriched/enrichment – isolation of fastidious bacteria – e.g. BHIA/BHIB differential – differentiate colonies (LF/NLF) and hemolytic pattern – e.g. negative = red negative NAFB = blue/green Describing a Bacteria GS reaction  cell shape  cell arrangement  spores 1. positive 2. negative a. McConkey agar – growth medium for gram (-) 1. cocci 2. bacilli 3. spirilla 1. diplo (pair) 2. tetrad 3. cluster 4. sarcina (8) 5. filament 6. singly – inhibitors of gram (+): bile salts, crystal violet – pH indicator: neutral red 1. central 2. terminal 3. subterminal Physiological Needs of Bacteria – positive: red, acidic, LF – 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, Strep. pyogenes, List. ivanovii obligate aerobes – has catalase and peroxidase that catabolize O 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. – e.g. a. potato/Sabouraud dextrose agar – fungal isolation b. thiosulfate-citrate-bile salt-sucrose agar – for Vibrio spp. 1. all Bacillus 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 – e.g. : skin penetration causes black Eschar 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 – e.g. India ink, nigrossin – uses 1. detects fungal meningitis from Cryptococcus neoformans – early morning sputum for sample – 3 successive negative results / days for clearance – causes 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 stained bacterial smear to be viewed in OIO 2. detects bacterial meningitis from Haemophilus influenzae major cause of meningitis in newborn, which is determined by hazy CSF and treated via lumbar tap – type a. low-grade fever esp. afternoon 3. Streptococcus pneumoniae obligate anaerobes – NO catalase and peroxidase – sediments growing in broth culture – use cork for closure 3. detects Bacilli (e.g. coliform) 1. C. perfringens – gangrenous legs of diabetic patients which are treated by hyperbaric oxygen 1. special stain 2. C. tetani – tennis racket bacilli that cause tetanus/lockjaw a. infectious particles – e.g. 3. C. botulinum – canned good bacilli that cause botox 4. C. difficile – cause of pseudomembranous colitis, 3 out of 4 General Microbiology (Lecture) 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