Microbiology Unit 1-6

Structure of Bacterial DNA and Plasmids

DNA- the most common macromolecule that encodes the genetic information and is composed of 2 strands of complementary nucleotides wound together in the form of a double helix. STRUCTURE: • Backbone of deoxyribose and phosphate groups • 4 nitrogenous bases: 2 purines-(A), (G) and 2 pyrimidines: (T), and (C) RNA- structurally similar to DNA except ribose sugar instead of deoxyribose and Uracil replaces thymine. TYPES: • Messenger RNA (mRNA) • Ribosomal RNA (rRNA) • Transfer RNA (tRNA) 》Genetic Diversity of Bacteria : 20mins spread quickly ▪︎Rapid reproduction ▪︎Mutation -errors/mistakes during DNA replication; change in nucleotide sequence of a gene, resulting from error during DNA replication TYPES OF MUTATION Spontaneous -without apparent cause Lethal -results in a death of cell; cannot be study or propagated in a lab Conditional -expressed only under certain environmental conditions Biochemical - Induced -chemical means or radiation treatments (ex. mutagens-heat or UV light) ▪︎Recombination - crossing-over and independent assortment 》GENE TRANSFER ▪︎Transformation -bacterial cell dies and undergo lysis; disintegration of the cell wall; can be transfer in a recepient bacterial cell ▪︎Transduction -transfer by a bacteriophage(virus that infects bacteria) transfer genes from one cell to another ▪︎Conjugation -transfer of genetuc material from 1 donor cell to a recepient cell PLASMIDS can be transfer through CONJUGATION: (HORIZONTAL TRANSFER) but not all plasmids are capable of conjugal transfer

Mycobacterium tuberculosis

slender, thread like, serpentine cord CORD FACTOR/TREHALOSE 6, 6'-dimycolate Red bacilli against blue/green background

Microbiology

study of all living organisms that are too small to be viewed by the unaided eye. ●Bacteria ●Viruses ●Fungi ●Prions ●Archaea ●Protozoa ●Algae

M. gordonae

aka "Tap water bacillus"; non-pathigenic; important contaminant of water' yields a false (+) staining reaction

Metabolism - Fermentation

an anaerobic process by which bacteria catabolize carbohydrates to produce energy.

Respiration (oxidation)

an efficient process by which obligate aerobes and facultative anaerobes generate energy

epistaxis

bleeding from the nose

pathogens

cause disease

petechiae

dengue rash

Normal flora /normal microbiota

do not cause disease

Gram's Variable: Mobiluncus spp.

found in human vagina; Gardnerella Vaginalis causing bacterial vaginosis Factors: 1. >24 hours of culture used 2. pH of the reagent used 3. Expiration date 4. Time over decolorize: G+ = G- under decolorize: G- = G+

Theodor Svedberg

inventor of ultracentrifuge

Acid fast stain step & function -acid fast stain reagent -acid fast organism -non-acid fast organism

primary dye/stain -carbol fuchsin (carbolic acid) -red -red ●CF -Fuchsia dye and 3-5% phenols/carboxylic acid Mordant/Accentuator -heat/tergitol -red -red Decolorizer/Differentiator -acid alcohol -red -colorless Secondary dye/ Counterstain -methylene blue/malachite green -red -blue/green

A. Discovery of Microbes

1. Lucretius with Girolamo Fracastoro, Roger Bacon and Anton van Plenciz ●Diseases were caused by INVISIBLE LIVING creatures 2. Francesco Stelluti ●Observed bees and weevils 3. Anton van Leeuwenhoek ●First true microbiologist; coined ANIMALCULES *First to describe bacterial morphology

2 general classifications of Microbiology

1. Acellular ●not true cells; obligate intracellular microorganisms; host dependent (viruses) 2. Cellular ●microorganisms consisting of cells; true cells (prokaryotic or eukaryotic)

Gram Stain General Rules:

1. All cocci are gram (+) except: Neisseria, Veilonella and Branhamella/Moraxella (Gram (-) cocci) 2. All bacilli are gram (-) except (17): Mycobacteria, Bacillus, Clostridium, Corynebacterium, Lactobacillus, Listeria, Erysipelothrix, Aerobic Actinomyces, Arcanobacterium, Gordonia, Rothia, Kurthia, Bifidobacterium, Propionibacterium, Fungus like Nocardia, Rhodococcus, Tsukamurella -Gram positive bacilli 3. Mycobacterium tuberculosis: are expected as gram positive however in actual staining it is gram ghost or gram neutral 4. Spirals/Spirochetes: are not stainable using gram staining however some are gram negative 5. Mycoplasma and Ureaplasma: are stained as gram negative: are cell wall deficient 6. Yeasts: are gram positive

B. Spontaneous generation /Abiogenesis

1. Aristotle ●simple vertebrates could arise from spontaneous generation 2. Francesco Redi ●Demonstrated that maggots did not generate spontaneously 3. John Needham ●Vital force; microbes developed spontaneously from fluids (boiled mutton broth) 4. Lazaro Spallanzani ●Improved Needham's experiment (broth sealed jar)

3 Main PARTS

1. Cell Wall ●important in serotyping: somatic O antigen (found in cell wall) Escherichia Coli - 181 O Ag Salmonella -more than 250 O Ag Shigella -A, B, C, D ●a. E. coli O 157:H7 :EHEC/Enterohemorrhagic E. coli -Hemorrhagic colitis/ Hemolytic Uremic Syndrome ●b. E. coli O111: EPEC/Enteropathogenic E. coli -infantile 2. Parts Lying Internal to cell wall 3. Parts Lying External to cell wall

Other Acid Fast Organism (AFO) [ with mycolic acid ]

1. Legionella micdadei 2. Rhodococcus 3. Nocardia -partial acid fast 4. Cryptosporodium parvum 5. Isospora belli 6. Cyclospora Cryptosporodium parvum, Isospora belli, Cyclospora - parasite

Parts Lying Internal to Cell Wall

1. Cytoplasmic Membrane -site for energy production/synthesis, semi permiable; maintains viability 2. Mesosomes -point of attachment for chromosome ●(serves as invagination during conjugation -transfer of genetic material from 1 bacterial cell to another cell ) 3. Free Ribosomes -site of protein synthesis 4. Inclusion Bodies -food and nutrient storage /reserve ●3 polyphosphate granules ●1. Much granules -M. tuberculosis ●2. Bipolar bodies -Yersinia pestis causative agent: bubonic plague/black death Wayson Stain: safety pin appearance: RED ●3. Babes-Ernst/Volutin/metachromatic granules -C. diphtheriae 5. Endospores /Bacterial Spores resting stage/hibernating -to maintain viability :heat resistant consist of dipicolinic acid and calcium ions =calcium Dipicolinate complex All are round/oval centrally located/subterminally located spores except: Clostridium tetani -round/oval but terminally located Clostridium botulinum -subterminally located Bacillus Anthracis -centrally located ●A. Bacillus -gram positive bacilli that can sporulate aerobically; catalase positive ●B. Clostridium -gram positive bacilli that can sporulate anaerobically; catalase negative

Parts Lying External to Cell Wall

1. Flagella/Flagellum -are whip like structure mainly for locomotion CHONS: Flagellin Antigen: H antigen (motile) "Hauch" H antigen for serotyping Yersinia, E. coli, Salmonella Types of Flagella A. Atrichous B. Monotrichous (Vibrio- monotrichous [ darting motility ]) C. Amphitrichous D. Lopotrichous -tuft of flagella on 1 side of bacterial cell E. Peritrichous -Enterobacteriaceae except for Klebsiella and Shigella (non motile) F. Bilophotrichous 2. Pili/pilus -are hair like projections CHONS: pilin TYPES OF PILI A. somatic/ ordinary/ common -adherance of bacteria to host cell; many in number and shorter B. Sex/ fertility -bacterial conjugation 3. Glycocalyx -serves as protection and for regulation A. capsule -organized material fiemly attached to cell wall; inhibit phagocytosis: easily cause MENINGITIS; bbb (break blood brain barrier) K Ag "Kapsule" = Vi Ag (virulent) salmonella typhi test: Quellung Reaction "swelling" 5 encapsulated 4 polysaccharide component of the capsule Neisseria meningitidis Haemophilus influenzae klebsiella pneumoniae steptococcus pneumoniae except: Bacillus anthracis -polypeptide D-glutamic acid b. slime layer -unorganized material not firmly attached to the cell wall Staphyloccus epidermidis: Normal flora of the skin -opportunistic pathogen -wound infection during open heart surgery

D. Theory of Antisepsis

1. Ignaz Semmelweis ●proper routine handwashing 2. Joseph Lister ●antiseptic surgery in britain

Functions of Cell Wall

1. It gives shape/ rigidity 2. Responsible for antigenic property 3. Pathogenicity ●A. M protein -streptococcus pyogenes M protein: inhibit phagocytosis: ICED Initiation, Chemotaxis, Engulfment, Digestion ●B. Mycolic Acid - Mycobacterium tuberculosis Mycolic acid/hydroxymethoxy -AFO/M. tuberculosis :resists digestion 4. Responsible for staining property

Points of Differentiation

1. Nuclear Body ●prokaryote: circular pattern (NUCLEOID) plasmids- are small, single, circular chromosomes of double stranded DNA arranged in circular pattern located in the nucleoid except: Borrelia burgdorferi /Borreliella burgdorferi ●eukaryote: arranged in linear pattern (NUCLEUS) Eukaryotic: DNA enclosed in a nucleus 2. Cell division ●pro: binary fission (asexual reproduction) ●eu: mitosis & meiosis (somatic /body cells: mitosis; sex cells: meiosis) 3. Cell wall ●pro: made up of peptidoglycan peptidoglycan layer /munein layer /mucopeptide layer: has an alternating backbone: N-acetyl glucosamine and N-acetyl muramic acid Without PG because they lack cell wall: Mycoplasm & Ureaplasma : pleomorphic (vary in shapes) Penicillin: beta-lactam drugs inhibit cell wall synthesis ●eu: animals/protozoa: no cell wall; plants/algae: present Cell wall of plants /algae: cellulose and glycan Fungi: glucan, mannan, chitin 4. Cytoplasmic membrane ●pro: membrane phospholipid bilayer without CHO and sterols except mycoplasma and ureaplasma: with sterols and CHO ●eu: fluid phospholipid with CHO and sterols 5. Site for CHON synthesis ●pro: free ribosomes ●eu: endoplasmic reticulum: Rough ER -attached firmly in the cytoplasmic membrane with ribosomes attached to it 6. Organelles ●pro: absent ●eu: present 7. Size ●pro: 70s -svedberg unit (unit of sedimentation); 50s & 30s ●eu: 80s - 40s & 60s 8. Energy/ATP synthesis ●pro: cytoplasmic membrane ●eu: mitochondria

E. Germ Theory of Disease

1. Robert Koch ●Germ theory; discovered anthrax bacillus, V. cholerae and Koch's bacillus; Koch's Postulate

C. Biogenesis - states that living cells can arise only from preexisting living cells

1. Rudolf Virchow ●concept of biogenesis 2. Louis Pasteur ●aseptic techniques - inhibit any form of contamination 3. John Tyndall ●tyndallyzation - sterilization technique; dust carry germs 4. Ferdinand Cohn ●described endospores-beat resistant bacterial structure 5. Antoine-Laurent Lavoisier ●oxygen significance to life

Bacterial Morphology

1. Shape 2. Arrangement -can be determine by plane of division COCCI ARRANGEMENT: spherical or round shape A. Bacterial cell that is determined by means of one (1) plane of division 》One (1) plane of Division ○cocci in pairs/diplococci: arrangement when cocci divide and remain together ○kidney/coffee bean shaped cocci (examples: Nesseria, Branhamella/Moraxella except for Neisseria elongata, Neisseria bacilliformis and Neisseria weaveri which are rod-shaped) ○Flame/lancet shaped: streptococcus pneumonia B. Bacterial cell that is determined by means of two (2) Plane of division 》Two (2) Plane of Division ○Packets of four/tetrads/square groups of four cells (example: Micrococcus tetragenus) C. Bacterial cell that is determined by means of three (3) Plane of division 》Three (3) plane of division in regular pattern ○Packets of eight (8) cells/cubical/sarcina/sarcinae (example: Micrococcus luteus) D. Bacterial cell that is determined by means of one (1) Plane of Division but continuously dividing 》One (1) plane of division but continuously dividing ○Cocci in chains: streptococcus: when cells adhere after repeated divisions in one place E. Bacterial cell that is determined by means of three (3) or more plane of division in irregular pattern 》Three (3) or more plane of division in irregular pattern ○Cluster of cocci: staphylococcus: when cocci divide in random planes BACILLI ARRANGEMENT: rod or elongated shape A. ○short plump/stout/small coccobacilli (example: Family Enterobacteriaceae B. ○Large square cut ends chain sporulated (example: bacillus anthracis) C. ○small, short bacilli, arranged in school of fish pattern (example: haemophilus ducreyi D. ○Large rounded end chains non sporeforming bacilli (example: bacteroides (anaerobe)) E. ○slender/threadlike/serpentine cord (example: mycobacterium tuberculosis (cording of bacilli is due to cord factor trehalose 6 6' -dimycolate)) F. some bacilli are arranged in chains (streptobacilli). Others are arranged at various angles to each other, resembling the letters 'V' presenting a cuneiform or chinese letter arrangement and is characteristic of Corynebacterium diphtheriae ○chinese letter or cuneiform (example: corynebacterium diphtheriae -pleomorphic, club shaped, fence stick, barbed, cigar packed, pallisade G. curved shaped, small -vibrio H. curved shaped, S, C shape, sea gull wing: campylobacter, helicobacter, arcobacter, -microaerophilic bacteria (2-10% O2) HELICAL/TWISTED ARRANGEMENT: A. SPIRALS ○Spirillum minor/minus B. SPIROCHETES: Genera: Borrelia, Treponema, Leptospira ○Borrelia: loosely twisted ○Treponema: lightly twisted resembling corkscrew ○Leptospira: tightly twisted but one or both ends are bent into a hook (leptospira interogans -causes leptospirosis; look interrogative mark)

Methods of Studying Bacteria

1. Staining- Staining Techniques ▪︎1. Special/Selective -stains specific bacterial structure ○a. Cell wall - Dyar, Dienes, Victoria blue dye ○b. Capsule - Welch, Anthony, Gin, Tyler, Muir, HISS ○c. Spores - Schaeffer-Fulton, Dorner, Wirtz-Conklin ○d. Spirochetes - Fontana Tribondeau, Levaditi Silver Impregnation, India Ink, and Warthin-Starry ○e. Flagella - Leifson, Gray, Fisher and Conn -> Tannic Acid: coat, swell, and precipitate ○f. Metachromatic Granules - Liubinsky, LAMB (Loeffler's Alkaline Methylene blue), Albert, Neisser, Burke's, Lindergran ○OTHERS: -Nucleic Acid: Feulgen, Acridine orange -Polar bodies: Wayson -Rickettsia: Gimenez stain, Macchiavello stain -Legionella: Dieterle silver stain -M. leprae: Fite-Faraco -Mycoplasma: Diene's stain -Negri bodies: Seller's stain ▪︎2. Indirect/Negative/Relief -employed to determine the morphology and cellular arrangement in bacteria that are too delicate to withstand heat fixing. It uses a dye solution in which the chromogen is acidic and carries a negative charge which then repels the negatively charged chromogen and so the bacterial cell remains unstained against a colored background; black backgroud while capsule is unstained ○Nigrosin and India Ink ○Anthony's stain ○Borris Method ▪︎3. Direct/Simple -use of a single or only one (1) stain or dye in a bacterial smear ○Crystal Violet, Gentian Violet, Eosin, Methylene Blue and Malachite Green ▪︎4. Differential Stain -the use of two (2) or more dyes or stains which divides or differentiates bacteria into separate groups ○a. Gram stain ○b. Acid fast stain ○c. Baumgarten -blue (M. tb) vs. red (Mycobacterium leprae "leprosy/Hansen's disease") ○d. Pappenheim's Stain -red (M. tb) vs. blue (M. laticola -formerly known as M. smegmatis) 2. Phenotyphic Characterization ○1. Cultural/Colonial ○2. Antigenic Determination ○3. Biochemical Reaction 3. Genotyphic Characterization -employs molecular and more specific immunological techniques 4. Animal Inoculation -uses standard McFarland 5. Motility ○a. Hanging Drop Technique/Wet preparation -vaseline Brownian Movement -LPO True Motility -Directional Darting Motility -vibrio Tumbling Motility -Listeria monocytogenes Gliding Motility -Capnocytophaga ○b. Semi-solid media (Sulfide Indole Motility (SIM) medium -incubated at room temperature (37°C); 0.5-1% of agar ○c. Flagellar stain

Kingdom Prokaryotes - unicellular

3 PHYLUM/PHYLA 1. Eubacteria -true bacteria (medically important bacteria) 2. Archaebacteria -primitive single celled (oldest organism living on earth) extreme temperatures (anaerobic) 3. Cyanobacteria -blue green algae; now blue green bacteria

Bacterial Cytology

70% -water 30% -CHO, CHONS, lipids & enzymes

Acid Fast Staining procedure

AFS -AFO VS NAFO Is a differential stain; not a confirmatory test TWO GENERAL METHODS: A. Ziehl-Neelsen /HOT Method ●ZN/HOT: 3g CF+ 5% phenol B. Kinyoun's /COLD Method ●K/COLD: 4g CF+ 9% phenol

SPECIMEN COLLECTION, TRANSPORTATION, HANDLING AND PROCESSING

Acute phase - Characterized by rapid onset of disease, a relatively brief period of symptoms. A. Basic principles of specimen collection ▪︎Collect the specimen in the acute phase of the infection and before antibiotics are administered. ▪︎Select the correct anatomic site for the collection of the specimen. ▪︎Collect the specimen using the proper technique and supplies with minimal contamination from normal microbiota. ▪︎Collect the specimen in a container or transport medium designed to maintain the viability of the organisms and avoid hazards that result from leakage. ▪︎Label the specimen accurately with the specific anatomic site and the patient identification -patient's name and unique identification number. ▪︎Transport the specimen to the laboratory promptly or make provisions to store the specimen in an environment that will not degrade the suspected organisms. ▪︎Notify the laboratory in advance if unusual pathogens or agents of bioterrorism are suspected. B. Collection procedures ▪︎Specimens for cultures must be collected in sterile containers except for stool specimen which can be collected in clean, leakproof containers. ▪︎Swabs are not recommended for routine collection. ▪︎Swabs are appropriately used for specimens from the upper respiratory tract, external ear, eye, and genital tract. ▪︎Tips of swabs may contain cotton, dacron, or calcium alginate. (cotton swabs may contain excessive fatty acids; two (2) swabs must be given -one (1) swab is for direct smear and the other one (1) swab is for culture ▪︎Wound specimen is collected from advancing margin of the lesion and should be collected by needle aspiration rather than by a swab. ▪︎Area should be cleansed to eliminate as much of the commensal flora as possible. C. Patient Collected Specimens ▪︎Provide verbal and written instructions. ▪︎Read the instructions to the patient. ▪︎Instructions should be written using simple language and pictures. ▪︎Most common specimens commonly obtained by the patient are urine, sputum and stool. ●Urine Collection ▪︎First morning clean-catch midstream urine specimen (catherized: aerobic; suprapubic aspirated urine: anaeroculture) Preservation: Boric Acid, Refrigerate 4°C =bacteriostatic (stool, sputum, swabs, and viral specimens), Carry/Cary blair: delay more than two >2hours Urine colony count: 10⁵ bacteria/ml or 1x1 10⁵ CFU/ml or > or = to 100,000 CFU/ml -UTI: 90% E. coli, Staphylococcus saphrophyticus: NF (sexually active young females) ●Sputum Collection ▪︎First early morning specimen ▪︎Expectorated sputum (three (3) consecutive, DOH: two (2) specimens/day ▪︎Induced sputum (patient inhales nebulized hypertonic solution =liquefying airway that produce sputum) Bartlett's Criteria 1. <10 epithelial cells/LPF : if >10 epith. cells (salivary) 2. >25 PMNs/LPF ●Stool Collection ▪︎Specimen of choice for the detection of gastrointestinal pathogens ▪︎Rectal swab can be submitted for bacterial culture as long as fecal material is visible on the swab ▪︎If bacterial infection is suspected, three specimens should be collected - one a day for 3 consecutive days ▪︎If parasite infection is suspected, three specimens collected within 10 days should be sufficient for microscopic detection of ova and parasites ▪︎Patient should be instructed to excrete directly into the collection device ▪︎Specimen should never be taken from the toilet and should not be contaminated with urine ●CSF -ultrafiltrate of blood (lumbar tap: third and fourth or fourth and fifth aspiration 3 three collection tubes: 1. Chemistry/serology -least affected by traumatic tap 2. Microbiology 3. Hematology (mostly affected by a traumatic tap) If possibly fourth 4 tube microbiology (for better exclusion of skin contamination) :only one (1) tube -ask for the priority test CSF: centrifuge to get for the sediment-------staining -India Ink and Gram Stain CSF Culture: BAP/CAP (maintain for 3days before diagnosed negative; examined daily) Bacterial Meningitis -capsulated 1. Birth to 1 month -Group B streptococci and Gram (-) rods 2. 1month to 5 yrs =H. influenzae 3. 5 yrs to 29 yrs: N. meningitidis 4. Over 29 yrs: S. pneumoniae 5. Listeria monocytogenes ●Genital specimens -venereal diseases: N. gonorrhea, C. trachomatis, M. hominis, U. urealyticum : these 4 can cause urethritis), (parasitic: Trichomonas vaginalis, Candida albicans: urethritis) ●Blood Culture: for fever of unknown origin (Leptospirosis, typhoid fever, brucellosis) collected -while the fever spikes (2 different sites) Anticoagulant: 0.025% SPS sodium polyanethol sulfonate ▪︎inhibit phagocytosis ▪︎neutralizes the complement activation and bactericidal effect of the human serum (antibody) ▪︎hemolyses aminoglycocide antibiotics 1. ARD -antibiotic removing device/antimicrobial removal device (ex. penicillin: penicillinase, Tetracycline: Magnesium sulfate, Sulfonamides: paramino benzoic 2. Thiol broth 3. Blood in TSB (Trypticase soy broth)-intended for aerobic culture: look for signs of bacterial growth (maintain for seven 7 days before declaring results: For suspected cases of Brucellosis- 3-4 weeks, For suspected cases of leptospirosis- 6-8 weeks) Signs of Bacterial growth: 1. turbidity 2. presence of bubbles 3. presence of blood clots 4. hemolysis ●Nasopharyngeal specimens a. Throat specimen -predominant pathogens: Group A streptococci/Streptococcus pyogenes Most predominant Normal Flora NF: Viridans streptococcus (alpha hemolytic streptococcus) b. Nasopharyngeal swab -(H. influenzae, N. meningitidis, B. pertussis-most significant) Group B Streptococcus/ Streptococcus agalactiae :commonly infect infant to 1month old for meningitis D. Labelling and Requisitions ▪︎Proper identification of each specimen includes a label firmly attached to the container with the following information: -Name -Identification no. -Room no. -Requesting Physician -Culture site -Date of collection -Time of collection ▪︎The requisition form should provide the following information: -Patient's name -Patient's age (DOB) and gender -Patient's room number or location -Physician's name and address -Specific anatomic site -Date and time of specimen collection -Clinical diagnosis or relevant patient history -Antimicrobial agents (if any) -Name of individual transcribing orders E. Preservation, Storage and Transport of Specimens ▪︎Specimen should be transported to the lab ideally within 30mins of collection, up to 2 hours. ▪︎Specimens such as urine, stool, sputum, swabs (not for anaerobes), foreign devices such as catheters, and viral specimens can be maintained at refrigerator temperature (4C) for 24hrs. ▪︎Pathogens that are cold sensitive can be kept in a room temperature if culture is to be performed ▪︎If CSF is not processed immediately, it can be stored in a 35C incubator for 6 hours

BACTERIAL METABOLISM

BACTERIAL METABOLISM- defined as the sum of all chemical processes that takes place in a living organism, resulting in growth, energy generation, waste disposal and other functions in relation to distribution of cell nutrients. 》Two major parts: Anabolism and Catabolism 1. Anabolism or constructive phase- there is synthesis of complex molecules and energy production. 2. Catabolism or destructive phase- large complex molecules are converted into simpler, smaller molecules accompanied by energy utilization.

Unit 2: Bacterial Cell Growth & Population

Bacterial Cell growth -simply defined as the proliferation or formation of new cells through replication During cell division via binary fission: one (1) parent cell forms two (2) progeny cells There is bacterial growth if there is an increase in the number of microbial cells in a population under favorable conditions Cell growth -referred to an increase in the number of microbial cells in a population Growth rate -defined as the change in the cell number or cell mass per unit time. Generation -the interval for the formation of two cells from one Doubling time/Generation time -time required for it to occur that is being calculated during the exponential phase of growth. Four Characteristic Phases of Microbial Growth Cycle in a Population/ Bacterial Growth Curve -these phases reflect the physiologic state of the organisms in the culture at that particular time 1. LAG Phase/period of rejuvenescence the period when microorganisms are introduced into fresh culture medium, usually no immediate increase in cell number occurs; the beginning of biosynthesis with no increase in cell mass and cell number noted. It is the adjustment period or adaptation period to a new environment. 》vigorous and increase metabolic activity; There is no apparent cell division occurring, the cells may be growing in volume or mass/increase in size; DNA and enzymes synthesis 2. LOG Phase/Logarithmic/Exponential Following the lag phase, the cells start dividing and their numbers increase exponentially or by geometric progression with time. If the logarithm of the viable count is plotted against time, a straight line will be obtained. It is the phase in which microorganisms are utilized in physiological and biological testing; susceptible or sensitive to antibiotics 》A growing bacterial population doubles at regular intervals. Growth is by geometric progression: 1, 2, 4, 8, 16, etc. or 2⁰, 2¹, 2², 2³, 2⁴ (where n=the number of generations) this is called exponential growth. 20mins= E. coli 24hours= M. tuberculosis 》is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression. The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation. 》Population Growth is limited by one (1) of the three (3) factors: -Exhaustion of available nutrients -Accumulation of inhibitory metabolites; toxic products -Exhaustion of biological space 3. Stationary/Plateau phase After a varying period of exponential growth, cell division stops due to depletion of nutrients and accumulation of toxic products. Eventually growth slows down, and the total bacterial cell number reaches a maximum and stabilizes. The number of progeny cells formed is just enough to replace the number of cells that die. The growth curve becomes horizontal. The viable count remains stationary as an equilibrium exists between the dying cells and the newly formed cells. 》Balance between cell dying = number of viable cells 》Secondary metabolites =end products in plateau phase 》Spore 4. Decline/Death Phase The death phase is the period when the population decreases due to cell death. Cell death may also be caused by autolysis besides nutrient deprivation and build-up of toxic wastes. The phase in which there is a loss of nutrients and increase in the amount of toxic products. 》cessation of bacterial growth

Branches of Microbiology

Bacteriology Mycology - molds & yeasts Parasitology Immunology Phycology - algae

Specimen Storage Temperature

E. Preservation, Storage and Transport of Specimens ▪︎Specimen should be transported to the lab ideally within 30mins of collection, up to 2 hours. ▪︎Specimens such as urine, stool, sputum, swabs (not for anaerobes), foreign devices such as catheters, and viral specimens can be maintained at refrigerator temperature (4C) for 24hrs. ▪︎Pathogens that are cold sensitive can be kept in a room temperature if culture is to be performed ▪︎If CSF is not processed immediately, it can be stored in a 35C incubator for 6 hours 》Table 4.1 Specimen Storage Temperature REFRIGERATE (4C) ▪︎Catheter tips (IV) ▪︎CSF for viruses ▪︎Ear: outer ▪︎Feces (unpreserved) ▪︎Feces for C. difficile toxin ▪︎Sputum ▪︎Urine (unpreserved) ▪︎Swabs ROOM TEMPERATURE (22C -25C) ▪︎Abscess, lesion, wound ▪︎Body fluids ▪︎CSF for Bacteria ▪︎Ear: Inner ▪︎Feces (preserved) ▪︎Genital ▪︎Nasal, throat ▪︎Tissue ▪︎Urine (preserved) FREEZER TEMPERATURE (-20C or -70C) ▪︎Sera (serological tests) (-20C) ▪︎Tissues (long storage period(-70C) ▪︎Feces for C. difficile toxin(-70C) if storage will be longer than 3 days Preservatives ▪︎Two specimen types in which preservatives can be used are urine and stool ▪︎Boric acid is used in commercial products to maintain accurate urine colony counts ▪︎Stool specimen for bacterial culture that are not transported immediately to the laboratory can be refrigerated ▪︎If the delay is longer than 2 hours, the specimen can be added Cary-Blair transport media. Anticoagulants ▪︎For blood, bone marrow and synovial fluid ▪︎SPS - most common anticoagulant used for microbiology specimens ▪︎EDTA and Citrate should not be used for microbiology specimens ▪︎Whole Blood (Heparinized)- ideal for virus isolation ▪︎Plasma (EDTA)- ideal for viral load/molecular tests and for serological tests

ENERGY PRODUCTION

ENERGY PRODUCTION-accomplished by the breakdown of chemical substrates through the degradative process of catabolism accompanied with oxidation-reduction reactions. Glucose is valuable nutrient for organisms TWO PROCESSES TO PRODUCE ENERGY FROM GLUCOSE A. Respiration- ATP generating process in which molecules are oxidized and the final electron acceptor is an organic molecule. Glucose is completely broken down; obligate/strict aerobes and facultative anaerobe Glucose ----CO2 and H2O Aerobic respiration- oxygen is the final electron acceptor Anaerobic respiration- different exogenous is the final electron acceptor. NITRATE, SULFATE, FUMARATE: final electron acceptor 1. Glycolysis (EMP) (Embden-Meyerhof-Parnas pathway)- first stage in carbohydrate metabolism. It is the oxidation of glucose to pyruvic acid. It is the major route of glucose metabolism in most cells. 2. Krebs Cycle (Tricarboxylic acid or TCA cycle)- it is the most important process for the complete oxidation of acetyl coenzyme A (CoA) substrate under aerobic conditions. It is an enzyme system which converts pyruvic acid to CO2 carbon dioxide in the presence of oxygen O2 with accompanying release of energy which is therefore trapped in the form of ATP molecules. B. Fermentation- It does not require O2 oxygen, the use of Krebs cycle or an electron transport chain. It uses an organic molecule as the final electron acceptor. ATP is generated only during glycolysis, thus only few molecules are produced. It releases energy from sugars or other organic molecules, such as amino acids and purines; mixture of end products: lactate, butyrate, ethanol, and acetoin 1. Alcoholic fermentation- ferment sugar into ethanol and carbon dioxide (for fungi, algae, protozoans and some bacteria). 2. Lactic acid fermentation- pyruvate is reduced to lactate: ○2.1 Homolactic fermentation- utilize glycolytic pathway and the enzyme lactate dehydrogenase directly reduce almost all the pyruvate to lactate. (for Streptococcus and Lactobacillus), which is used to make yogurt, pickles and sauerkraut. ○2.2 Heterolactic fermentation- process producing substances other than lactate such as alcohol, carbon dioxide, formic acid, and acetic acid. 3. Formic acid fermentation- pyruvate is converted to formic acid (Enterobacteriaceae) ○3.1 Mixed acid fermentation- the production of ethanol and acids such as lactic, acetic, succinic and formic acid.; ethanol and mixed acids ○3.2 Butanediol fermentation- pyruvate is converted to acetoin and afterwards reduced to 2,3- butanediol with NADH, small amount of ethanol and mixed acids are also synthesized; butanediol fermentation: acetoin ENERGY UTILIZATION Once energy is obtained, bacteria as well as other organisms utilized it in various ways such as: o For the biosynthesis of new cell components o For the maintenance of physical and chemical integrity of cell o For the activity of the locomotor organelles o For the transport of solute across membranes o For heat production

BACTERIAL GROWTH (Nutritional and Environmental Requirements)

Factors that influence the rate of bacterial growth: Nutritional, Environmental or physiologic, and Essential 1. NUTRITIONAL ●a. Nitrogen - for protein synthesis ●b. Carbon - main constituent of cellular materials; for bacterial cellular elements synthesis Classification as to carbon source: AUTOTROPHS -use inorganic compounds as a sole source of carbon (ex: carbon dioxide CO²) HETEROTROPHS -use organic compounds as source of carbon (ex. CHO and lipids) Classification as to electron source: LITHOTROPHS -uses inorganic chemicals as energy source ORGANOTROPHS -uses organic chemicals as energy source ●c. Water/Moisture ●d. Additional requirements/additives (vitamins or growth factor -fastidious organisms ex. haemophilus: require both X and V factor Factor X and V =blood Factor X/Hematin/hemin -degradation product of hemoglobin; heat stable Factor V/NAD/Nicotinamide adenine dinucleotide/NAD Coenzyme/Coenzyme; heat labile 》On BAP (Blood Agar Plate) -X factor; has enzyme that inhibits V factor 》On CAC (Chocolate Agar Plate) -when you reheat the BAP at 60-80C, the enzyme will be denatured = lysis of RBC; CAP contains both X and V factor; the primary isolation culture media of choice for haemophilus 》Other sources of V factor: potato yeast extract staphylococcus aureus ●e. Mineral ions- Iron, Magnesium, Calcium and Potassium and trace elements ●f. Salt/NaCl - for halophilic organisms - 5-6% of salt concentration. More than 6% of salt/NaCl = inhibitory 》Enterococcus and Vibrio cholera Haloduric: S. aureus: at Mannitol Salt Agar/ Chapman Agar (7.5% salt inhibitor) 2. ENVIRONMENTAL or physiologic ●a. Gaseous/Oxygen Requirement ▪︎1. AEROBE - bacteria can live, grow and survive in the presence of oxygen. +O2 ○1.1 STRICT/OBLIGATE AEROBE - absolutely require oxygen to grow. Examples: Pseudomonas, Mycobacteria, Brucella, Bordetella, Neisseria, Leptospira and Francisella ○1.2 FACULTATIVE ANAEROBE - does not strictly require oxygen but grow is better in the presence of oxygen. Examples: Enterobacteriaceae, Staphylococci, Streptococci 》Note: Both 1.1 and 1.2 have protective enzymes against oxidation 》Toxic products of oxidation: Hydrogen Peroxide H2O2 = catalase (enzyme) H2O2 -----catalase---- =H2O + O2 Ozone O3 - SOD superoxide dismutase O3 ------- SOD -------- O2 + O2 ▪︎2. ANAEROBE - bacteria can live, grow and survive without the presence of oxygen. ○2.1 STRICT/OBLIGATE ANAEROBE - absolutely does not require oxygen to grow. Example: Clostridium and Bacteroides ○2.2 AEROTOLERANT ANAEROBE - are technically anaerobe but it can survive even with the presence of oxygen; SOD but no catalase Examples: Enterococcus, Lactobacillus and Propionibacterium MICROAEROPHILIC - grow under reduced O2 (5-10%) and increased CO2 (8-10%) (2-10%) concentration of oxygen O2 CHA: Campylobacter, Helicobacter and Arcobacter CAPNOPHILIC- requires 5-10% of CO2; also, strict aerobe (15% O2 and 0.03% CO2 : uses candle jar (white candle) Neisseria, Haemophilus, Aggregatibacter now Actinobacillus, Cardiobacterium, Eikenella, Kingella and Streptococcus pneumoniae ●b. Temperature - 35 C-37 C optimum temperature <20C - Psychrophilic/Cryophilic 30C - 37C - Mesophilic 40C-45C - Thermophilic 80C-110C - Hyperthermophile/Extremely Thermophilic 10-37-45C - wide/long range - Eurithermophilic (ex. Enterococcus) 35-37C - narrow/short range - Stenothermophilic (ex. Neisseria) ●c. measure of hydrogen ion concentration Optimum pH: 7.0-7.5 (neutral or slight alkaline pH <3.0: Acidophile/Acidophilic (ex. Lactobacillus acidophilus: normal flora of vagina and can be found in mouth Medium: Tomato Juice Agar (TJA) pH 8-10: Alkaliphile (ex. Vibrio spp.) Medium: Alkaline Peptone Water (APW): enrichment culture media

Unit 6: Sterilization and Disinfection

Joseph Lister -antiseptic procedure Ignaz semmelweis -handwashing Terminologies ●Sterilization: complete destruction of all microbial cells (all forms of life: pathogens, non-pathogens, vegetative cells, bacterial spores, viruses, and prions) STERILIZATION is the complete killing and removal of all living microorganisms including bacterial spores. ●Disinfectant: deals with destruction of pathogens; does not remove bacterial spores DISINFECTION refers to the removal, inhibition, or killing ofmicroorganisms which include potential pathogens, by using chemical agents usually on inanimate objects, although it does not remove all bacterial spores. THERMAL DEATH TIME (TDT) - is the minimum time required to kill a suspension of organism at a predetermined temperature in a specified environment. THERMAL DEATH TIME (TDT) - is the minimum time required to kill a suspension of organism at a predetermined temperature in a specified environment. FACTORS THAT INFLUENCE THE DEGREE OF KILLING a. Type of organism b. Size of inoculum c. Concentration of disinfecting agent d. Nature of surface tobe disinfected e. Contact time - 1-2minutes f. Temperature - directly proportional to the time needed to kill bacteria/disinfection process g. pH -acid is better disinfectant h. Biofilm formation i. Compatibility of disinfectant and sterilant

UNIT 5: MICROBIAL CONTROL AND ANTIMICROBIAL AGENTS

Microbial control - aims to inhibit and prevent bacterial growth by involving the use of physical and chemical agents. Microbes are controlled in either of the two basic ways: (1) by inhibiting the microbial growth or (2) by killing the microorganisms. antimicrobial - Agents that kill the microorganisms or inhibit microbial growth antibiotics - are the substance produced by microorganisms to selectively suppress the growth or kill the microorganisms at its lowest concentration possible. Antibiotics can also be produced through chemical procedures that are independent from microbial activity. ANTIMICROBIAL AGENTS - can reduce or destroy undesirable microbes in a given area through killing and/or inhibiting. Its action can be done using - physical, chemical and mechanical method or agent. Characteristics of antimicrobial agent: 1. Inactive form 2. must be able to achieve concentration that is higher that the pathogen's MIC (minimal inhibitory concentration) 3. Must have selective toxicity 2 classes of antimicrobial agents: 1. Antibiotics -metabolic products (Other sources aside from bacteria: Fungi, molds, actinomycetes) 2. Antimicrobial Chemotherapeutic chemicals -chemical synthesized in the laboratory Mode of Actions of antimicrobial agents: 1. Bacteriostatic -inhibit the growth of cells without killing them; keep them in the stationary phase; interferes witj bacterial CHONS (ex. chloramphenicol, dapsone, erythromycin, clindamycin, isoniazid, rifampicin, sulfonamides, tetracycline-inhibit ribosome, trimethoprim and sulfamthoxazole 2. Bactericidal -kills bacterial cells; used to treat lofe threathening infections; inhibit cell wall; inhibit bacterial enzymes or CHONs translation (ex. aminoglycosides: gentamicin, amikacin and streptomycin Beta-lactam: penicillin, ceftriazone, imepenem and cefotaxime Glycopeptides: vancomycin, isoniazid, quinolones, bacitracin and metronidazole) 3. Bacteriolytic -act to lyse the bacteria by decreasing the total cell count and viable cell count ANTIBIOTICS 1. Narrow Spectrum antibiotics -targets few types of bacteria 2. Broad spectrum antibiotics -many types of bacteria ▪︎both narrow and broad use to treat infections CLASSIFICATION OF ANTIBACTERIAL DRUGS/ANTIBIOTICS 1. Natural- produced by bacteria or fungi (ex. Beta lactam-targets Cell wall synthesis: penicillins, cephalosporins: cephalosporium-treat gonorrhea, and cephamycins) ▪︎Other natural drugs: streptomycin, nystatin, rifampicin, tetracycline, vancomycin, bacitracin, gentamicin, and polymyxin 2. Semi-synthetic- are modified natural drugs with added chemical groups (ex. ampicillin, methicillin and carbenicillin) 3. Synthetic- are chemically produced drugs ▪︎Paul Ehrlich studied selective toxicity -discovered treatment for syphilis: salvarsan ▪︎Sulfa drug- Gerhard Domagk (sulfanilamide: simplest form of sulfa drug) ▪︎Isoniazid -for Mycobacterium; interferes with mycolic acid synthesis ▪︎Nucleic acid base analogs -added bromine and fluorine ▪︎Quinolones -interfere DNA gyrase (ex. ciprofloxacin)

Phenol Coefficient

Phenol Coefficient -expression of the bactericidal power of a particular substance or agent as compared to pure phenol FORMULA PC=highest dilution of disinfectant that will kill organism at a given time/highest dilution of phenol that will kill organism at a given time RESULT: PC >1 indicates that the disinfectant is more effective than phenol Interpretation: The higher the PC value, the more effective the disinfectant

Measurement of Growth

Population growth is measured by following changes in the number of cells or weight of cell mass. Direct cell count: Total count (both dead and living) and/or viable count (only living cells are counted) Different Methods for the Measurement of Cell Mass: 1. Direct Techniques 》Direct Physical: Dry Weight and Wet Weight 》Direct Chemical: Total Nitrogen, Total Protein and Total DNA content 2. Indirect Techniques 》Rate of O2 production or consumption 》Rate of CO2 production or consumption 》Turbidimetry -uses colorimeter Methods for the Measurement of Cell Numbers: 1. Direct Cell Count 》Counting chamber: Petroff-Hausser counting chamber 》Electronic counter: Coulter counter 》Membrane filter 2. Indirect viable cell count 》Plating method The usual way to perform a viable count is to determine the number of cells in the sample capable of forming colonies on a suitable agar medium. There are two ways of performing a plate count: spread plate method pour plate method In either case the sample must usually be diluted before plating. 》Membrane filtration Microbial cell numbers are frequently determined using special membrane filters possessing Millipore small enough to trap bacteria. In this technique a water sample containing microbial cells passed through the filter. The filter is then placed on solid agar medium or on a pad soaked with nutrient broth (liquid medium) and incubated until each cell develops into a separate colony. Membranes with different pore sizes are used to trap different microorganisms. Incubation times for membranes also vary with medium and the microorganism. A colony count gives the number of microorganisms in the filtered sample, and specific media can be used to select for specific microorganisms. This technique is especially useful in analyzing aquatic samples.

Gram Stain step & function- gram stain reagent -gram positive bacteria -gram negative bacteria

Primary dye stain -crystal violet -purple -purple Mordant/Accentuator -Gram's Iodine -purple -purple Decolorizer/Differentiator -Acetone +95% ethanol -purple -colorless Secondary dye/counterstain -safranin o/safranin red -purple -red/pink

Synthesis of Polypeptides

TRANSCRIPTION to TRANSLATION

Gene Transfer

TRANSFORMATION TRANSDUCTION LYSOGENIC CONVERSION CONJUGATION 1.Transcription- is the synthesis of single-stranded RNA using one strand of the DNA as a template. This converts the DNA base sequence of the gene into an mRNA molecule that is complementary to the gene's DNA sequence. 2. Translation- is the actual synthesis of as specific protein from the mRNA. 3. Transformation- naked DNA is taken up from the environment by bacterial cells. It involves the recipient cells up taking free DNA that is released into the environment when another bacterial cell (donor) dies and undergoes lysis or cell disintegration. 4. Transduction- a second mechanism by which DNA from two bacteria may come together in one cell, thus, allowing for recombination. This process is mediated by viruses that infect bacteria (bacteriophage). 5. Conjugation- the third mechanism of DNA exchange between bacteria. It is a direct transfer of DNA from one bacterial cell to another. 6. Mutation- is a change in the original nucleotide sequence of a gene or genes within an organism's genome. 7. Recombination- a process by which genes are transferred or exchanged between homologous regions on two DNA molecules.

Microbes /microorganisms

microscopic forms of life -disease causing agents

Gram Stain /Staining by Hans Christian Gram

most commonly used differential staining technique which differentiates 2 large groups of bacteria; the gram positive and the gram negative group of bacteria G+ violet/purple G- red/pink

Kingdom Eukaryotes - cells that contain nucleus & organelles enclosed by a plasma membrane

●Animals, plants, fungi, protozoa, algae

Unit 3: Bacterial Genetics and Metabolism

》Bacterial Genetics -genetic material must be accurately duplicated -passed on to generations -ensure normal function Genetics - concerned in the study of heredity and variation of inherited traits or characteristics. Nuclei acid structure and organization consists of two major classes- the DNA and RNA. DNA is a double-stranded helical chain of nucleotides/double helix/spiral staircase that encode the genetic information 》DNA- deoxyribonucleic acid (the most common macromolecule that encodes genetic information) 》DNA consists of deoxyribose sugars connected by phosphodiester bonds. The bases that are covalently linked to each deoxyribose sugar are the key to the genetic code within the DNA molecule. 》Complementary base pairing: A-T RNA: A-U (Uracil) G-C two (2) purine: (A and G) two (2) pyrimidine: (T and C) Molecular Structure of DNA 》Each Strand has 5' to 3' hydroxyl terminus 》Antiparallel 》"Twisted ladder" 》In contrast to DNA, RNA rarely 》exists as a double-stranded molecule Nucleotide -building blocks of nucleic acid >one (1) single unit of nucleotide -(monomer)- sugar, phosphate, base >DNA and RNA -nucleotide polymers RNA is a single-stranded and short nucleic acid containing ribose where in some forms, they encode genetic information for some viruses. 》RNA- ribonucleic acid (in some forms, RNA encodes genetic information for various viruses) 》THREE (3) MAJOR TYPES OF RNA ▪︎mRNA -blueprint for protein construction ▪︎rRNA -exactly where protein synthesis occurs. subunits that forms 70s ribosomes ▪︎tRNA -tranfers amino acids to ribosomes for protein synthesis Flow of Genetic Information ▪︎Replication -duplication of chromosomal DNA for insertion into a daughter cell; 2 progeny cells gets the full and exact genetic combinant from the original parent cell 40mins in a rapid growing bacteria Gene expression: polypeptide synthesis ▪︎Transcription -synthesis of a single stranded DNA as a template; Converts DNA base sequence into mRNA. RNA polymerase speeds up ▪︎Translation -genetic code with mRNA translated to a specific amino acid sequence that codes for specific protein on a ribosome INITIATION -codon A U G ELONGATION -ribosomes moves along the mRNA TERMINATION -stop codon reached Transcription & Translation -crucial to protein synthesis conjugation -the genetic expression involves various processes and mechanisms for gene transfer from one bacterial cell to another recipient cell Bacterial metabolism consists of biochemical reactions, the sum of all chemical reactions that occur in a living organism for their growth, energy synthesis, waste disposal and excretion and other related functions for nutrient distribution among cells. 》Genes and the Genetic Code ▪︎A DNA sequence that encodes for a specific product (RNA or protein) is defined as a GENE ▪︎All genes taken together within an organism comprise that organism's GENOME ▪︎The genome is organized into discrete elements known as CHROMOSOMES Genetics- the study of heredity and variation Gene- the unit of hereditary; a segment of DNA that specifies for a particular polypeptide Genome- the sum total of all the genetic information contained in a cell

Other scientists

》Robert Hooke ●cell theory 》Fanny Hesse ●use of agar as solidifying agent 》Julius Richard Petri ●petri dish 》Edward Jenner ●concept of vaccination 》Emil von Behring ●Antitoxins for diphtheria and tetanus 》Elie Metchnikoff ●Phagocytosis 》Selman Waksman ●Father of antibiotics - streptomycin and neomycin 》Alexander Fleming ●Penicillin (Penicillium notatum); lysozyme 》Paul Ehrlich ●Salvarsan (arsphenamine) for syphilis treatment