Lab definitions:

Peptone

Fastidious

Axenic

p: short peptide units, fragments of amino acids

f: picky, may not grow without an enrichment

a: pure culture

Match physiology with medium thats appropriate in order to be cultured

- some species cannot be cultured because the medium needed is unknown

Photolithotrophic

Chemolithotrophic

Chemoorganotrophic

p: photosynthetic, source of reducible carbon, energy of sunlight to ATP

cl: inorganic environment, ferrous sulfate

co: chemoheterotrophic, organic source of carbon, organic substrate for ATP yielding oxidation reactions

- things that spoil (vegetables, fruit, milk)

- human body, water, soil

- Pasteur used a sliced potato

1. Synthetic/chemically defined- ideal, but expensive, time consuming and difficult- exact amounts

2. Non-synthetic- micro lab, exact formulation unknown

3. Complex- include extra substances, fastidious substances will grow

convert liquid medium to solid

seaweed/kelp extract, frequent solidifying agent

s: designed to allow the growth of the desire microorganism one is trying to isolate and suppress the growth of others (whats added or left out)

d: distinguish between two types of growth on a plate; phyical appearence of colonies when cultured (pH indicator, fermentable carbohydrate)

m: selective medium, bile salts and crystal violet are incorporated to inhibit growth of some bacteria and permitting others

b: differential medium, erythrocyte effects

- gamma = no effect, alpha = destruction, beta = complete breakdown

1. tryptic soy agar/broth- wide variety of bacteria (all purpose), chemooranotrophs

2. eosin methylene blue agar- dyes as indicators for differentiation that ferment lactose/produce acid

3. phenylethyl alcohol agar- select growth of a large group of bacteria (limits g-)

4. macconkeys agar- intestinal bacteria, ferment lactose (gray vs. red)

1. proper amounts of each ingredient must be dissolved in water

2. check pH/adjust

3. medium dispense into containers

4. sterilized in autoclave or by filtration (121 degrees), can kill endospores

use inoculating loop to distribute bacteria into 3 regions of petri dish

- top to bottom, right to left, bottom to top

- sterilize after each region

- separate components of a mixture

mark on bottom:

medium, bacteria, group identifier, date, incubation temperature

1. Serratia marcescens

2. Staphylococcus epidermidis

1. g-, bacillus, broth, nosocomial infection (urinary or GI)

2. g+, coccal, broth, skin flora

- experimental atmosphere

- different atmospheric conditions can be established

- used in labs that cannot afford glove boxes

acidophiles- grow at pH of 0 or 1, below 5.5

alkalophiles- grow at pH of above 8.5

neutrophiles- grow at pH between 6.5 and 7.5 * majority of disease causing bacteria

Lining of the stomach- flagella to move

grows in acidic condition

helical shape

g-

1. biomass measurements

2. direct cell counts

3. viable direct cell counts

- type of biomass measurement

- taken by spectrophotometer or klett sommerson photometer

- single wavelength of light (monochromatic) neturalizes yellow medium at 540 nm

- registered on photo electric cell

- read at 100% transmission

- % transmission decreases as more light comes through

- medium becomes darker color

advantage: easy to do

disadvantage: does not distinguish between living and dead cells

- Petroff-Hauser counting chamber

- Hemocytometer- blood cell count

- Coulter counter/FACS machine

- DMC- direct microscopic count/breed smear

** all do not distinguish between viable and dead cells

- AKA particle counter

- thin capillary tube, electric current, electrical detector, automatic counter

- 2/10 of a mL sample size

- can set window/gate of what cells to be counted within a size range

- FACS= fluorescence activated cell sorter

- standard plate count (SPC)- measure of formation of colony (most accurate method)

- colony forming units/mL on plates- ableto divide and form a population of identical cells

- growth takes time, not immediate results

- plate count x dilution factor

- absorbance graph/standard curve can show # of viable cells

- MPN- most probable number, water quality

p: filtration, heat/temperature, irradiation

c: anitbiotics, chemotherapeutic

- destruction or removal of all life forms, microbial or otherwise in or on a product referred to as sterile after

 - done by heat, irradiation, filtration, selected chemicals

- physical means are usually more reliable or absolute than chemical measures

- the reduction of the number of organisms (with or without sterilization) to a level that is not harmful to the health of a living animal or the quality of perishable goods

 - destruction of vegetative cells but rarely spores

 - treatment of inani-mate surfaces and objects

- poisoning or breakdown of living tissue due to the growth of microorganisms or the presence of their products

- usually manifests itself as the formation of pus

- chemical agent used in the destruction or inhibition of microbial growth on living tissue

- limiting or preventing the harmful effects of infection

- reduction of the number of microorganisms to an acceptable level

- dictated by local public health agencies

- static means to inhibit

- cidal means to kill

 compound that is used for the treatment of infections/neoplasia in man or animals

compound that is used for the prevention of infection in man or animals

- ex) the administration of quinones and quinone when entering areas indigenous with malaria

- food- Campylocacter & Salmonella spp.

- blood/blood products

- endospore- Bacillus/Clostridium spp.

- Staphylococcus spp. (aureus)

- Mycobacterium spp.- acid fast/mycolic acid makes more resistant

- Pseudomonas spp. (aeruginosa)

- fungal cells- dermatophytes

- viruses- naked or enveloped

cloudiness of a fluid (broth) caused by individual particles

- asses amount of microbial growth

- why microbiologists need special instruments to measure growth

* colorimeter or spectrophotometer at 500-700 nanometers

1. type of organism expected

2. microbial load

3. environment organisms are found

4. intended use of the material/situational considerations

- number of organisms (present/anticiapted)

- logarithmic growth

- best temperature, time length, and medium for 90% of population to become inactivated

- time it takes for a population to become inactivated, very predictable, logarithmic

- longer to inactivate larger population

- decrease by 1 log at a time (10^8 to 10^7)

1. temperature (ex. E. coli at 70 degrees has smaller d value than 50 degrees)

2. genera of bacteria (heat sensitive is closest to axis, endospores farthest)

3. foodborne pathogens (Staph. aureus has largest d value)

food production

- in order to prove sterilization

- prescence of organic products (carbohydrates and fats)- more present, longer time to treat it

- temperature, pH and extraneous environment

1. dry heat- incineration or hot air oven

2. moist heat- boiling water, pasteurization, or heat with pressure

- both dry heat

i: place into flame (bunsen burner, benchtop incinerator)

h: 160-170 degrees for 2 hours, will inactivate endospores

- will kill vegetative cells in 5 minutes at 70 degrees C

- inactivate enveloped viruses and fungi

- not a sterilant because does not inactivate endospores and naked viruses

- differential sensitivity- 30 minute exposure maximum

- developed by Pasteur for beer

- used to prevent Mycobacterium bovis in milk

- Coxiella burnetti is controlled by pasteurization- can cause respiratory disease

1. Batch- LTLT- use vat at 62 degrees C for 15 minutes

2. Flash- HTST- tubes at 72 degrees C for 15 seconds **milk in grocery stores

3. ultrapasteurization- UHT- ultra high temperature, sterile, not refrigerated, 142 degrees C for 3-4 seconds

- autoclave/pressure cooker

- considered to be sterile

- saves time

- minimum of 15 minutes at 121 degrees C with 1 additional atm for 1 tube

- more volume, more time- takes longer to penetrate the center (ex. molasses)

gram +: limited growth with MAC and EMB so PEA promotes growth

gram -: limited growth with PEA so MAC and EMB promote growth

- general all purpose growth medium

- form, elevation and margin

- elevation is 3D and seen with stereomicroscope

- color and size

- light passage

mannitol salts agar

- 7.5% NaCl (high concentraion)

- inhibitory to everything but Staphylococcus spp.

- can tolerate salt, turns yellow

- selective and differential

MAC- fermenter is red, non-fermenter is white

EMB- fermenter is metallic green

type of blood agar with 5% blood cells

- 3 types of hemolysins:

* alpha- incomplete, viridans

* beta- complete, strep throat

* gamma- ahemolytic, without

- classify Staphylococcus & Streptococcus spp. with agar

- selective to differential medium

- important with antibiotics and chemotherapeutic drugs

- first chemotherapetuic therapist

- worked with Treponema pallidum

- arsphenamine/compound 606/salvarsan- chemical containing arsenic that was effective in treating syphillis

- important with antibiotics and chemotherapeutic agents

- second golden age of microbiology

- work led to penicillin discovery

- discovery of lysozymes

- mold growing on Staphylococcus auerus helped penicillin discovery

- zone of inhibition- cells died

a: chemical produced by certain molds and bacteria that kills or inhibits the growth of other microorganisms

ca: chemical used as a therapeutic medication to treat a disease

a: Streptomyces spp., Penicillium notatum/chrysogenes, soil organisms

c: compound 606

1. antibiotics- limit competition in environment with limited resources

2. Synthetic- man made in laboratory

3. Semi-synthetic- antibiotics produced by microorganisms then altered by organic chemist

1. streptomyces spp.- streptomycin, tetracycline

2. bacillus spp.- bacitracin, polymyxin

3. penicillium spp.- G and V

synthetic: chloramphenicol, sulfonamides

semi synthetic: penicillin derivatives- ampicillin and methicillin

- cidal/static/removal

- spectrum of activity

- mode of action

classification base on effect on "target"

- cidal = kill pathogen

- static = inhibit pathogen

- removal- depends on immune system of host

classification based on effect on "target"

- broad- comprehensive treatment when infectious agent is unknown (mycobacterium, g+ and g-)

- narrow- select group; leaves normal bacteria flora intact (only mycobacterium, only g-)

Narrow mycobacterium exmaples (2)

Narrow spectrum g- explanation

Broad examples (2)

- mycobacterium: isoniazid- tuberculosis, dapsone- leprosy

- G- is narrow because it effects treponema, penicillin and neisseria only

- broad: tetracycline and chloramphenicol

classification based on effect on "target"

- inhibit peptidoglycan synthesis

- inhibit unique prokaryotic metabolic/enzymatic pathway

- protein synthesis inhibitors

- inhibit membrane function/destroy membrane activity/selectivity

- interfere with nucleic acid synthesis

- penicillin

- cephalosporin

- vancomycin

- bacitracin

sulfonamides

-- sulfa drugs --

- aminoglycosides

- tetracycline

- erythromycin

** act on ribosomes **

- polymyxin

- amphotericin B

- nystatin

rifamycin- RNA transcription

nalidxic acid- DNA replication

- does not induce drug allergy in host/patient

- does not induce drug resistance in target organism/pathogen

- exhibits high selective toxicity

- allergy: 10% of population allergic to penicllin

- resistance: major problem today with control of disease (ex. MRSA- methicillin, vancomycin)

- selective toxicity: high therapeutic ratio measured by chemotherapeutic index (ex. Pseudomonas- nosocomial/hospitcal acquired)

- Paul Ehrlich

- formula: chemotherapeutic index = lowest dose toxic to patient / minimal curative dose

- minimal curative dose inhibits or kills pathogen

- comparative biochemistry

- exploiting differences in basic biochemical processes between bacterial and mammalian cells

- selectively exploit the difference in biochemistry between different microbial organisms

- comparative cytology

- selective toxicity

- exploit the differences in prokaryotic cell structure

 - knowledge of cellular staining characterisitics of different organisms is the first step to understanding toxicity

- buy time so the immune system can rid of the microbial invader

- recovery if immune system beats organism due to innate resistance or acquire immunity

- disease due to strong virulence mechanism

- unforeseen consequences

- drug interactions- consider on individual basis

- good outcome if compund is -cidal/-static

- bad outcomes: metabolize, metabolizing inactivated, allergy, resistance

- major means of control

- highly favorable chemotherapeutic index

- not without problems (ex. hypersensitivity - allergy)

basic penicillin

- all penicillins and cephalosporins are beta-lactams

- characteristic beta-lactam ring in every beta-lactam organism

- thiazolidine ring stabilizes beta-lactam ring but is substitued for different penicillins

- increase resistance to beta-lactamases and penicillinases

- increase spectrum of activity (ampicillin carbenicillin)

- different class but still produced by fungi

- after penicillin

- beta lactam ring present

- dihydrothiazine ring instead of thiazolidine

- relatively more resistant to beta-lactamases

- exhibit wider spectrum of activity

- not as likely to produce allergy

- improved pharmacokinetics - more efficient in body

- penicillin binding proteins (PBP)

- responsible for catalyzing reaction

- prevent transpeptidation

- many different ones in a cell

- involved in cell division and growth

- PBP important to interbridge and side chain

- bactericidal in rapidly growing/dividing cells

- structural analogy between penicillin and terminal d-alanine-d-alanine amino acids of the side chain

- similarities in structure can confuse PBP and it will bind and not reverse

- typically catalyzes where interbridge connects to 1 d-alanine

- beta lactams inhibit catalyzation

- beta lactamases cause resistance

- clavulanic acid has beta ring; AKA "suicide inhibitor" of beta-lactamases

- beta-lactamase inhibitor works by forming a stable complex with beta-lactamases

- extend spectra of some penicillins

- amoxicillin, clavamox

- complex, multistep process

- large number of enzymes, transporters, structural and membranal proteins

- intracellular and extracellular stages

- no ring, but are still cell wall inhibitors

- prevent monomer release: bacitracin, d-cycloserine

- prevent side chain from forming (block growth point): vancomycin, ristocetin

isoniazid

- similar to molecule that inhibits mycolic acid synthesis

- inhibits mycobacterium/nocardia

- used in combination with ethambutol or pyrazinamide for tuberculosis treatment to prevent resistance

- class of drugs

- sulfonamide is an example

- prevent metabolic/enzymatic pathway

- unique to prokaryotes

- chemotherapeutic not antibiotic

- common ring structure

- enhance effectiveness of inhibtion & change pharmacokinetics

- enzymes fit in particular active site based on shape

- inhibition by similar shaped organism taking up active site

- less affinity

- prevent enzyme from catalyzing appropriate reaction (enzymes fooled- form dysfunctional product)

- leads to more discrimination by enzymes, increasing chance of resistance

-dihydropteridine, PABA, and glytamic acid synthesized by dihydropteroate synthetase *critical

- dihydrofolate is formed, synthesized by dihydrofolate reductase

- forms tetrahydroflorate (folic acid)

similar structure (structural analogs)

- competitive with each other to reach active site since similar shape

- higher ratio of sulfanomide inhibits PABA attachment

- PABA has SO2 & NH2 groups, sulf has COOH

- vary in size, shape, number, location and composition

- composed of polymetaphosphate

- AKA volutin granules

- Burdon's method

- PHB: poly-b-hydroxybutyrate is revealed

- heat fixed smear, sudan black B for 15 min, xylene for 10-20 sec, counterstain 5-10 sec with .5% safranin, first water wash

- lipid deposits are black, rest of cell is red

- Schaeffer fulton method

- heat fixed smear, saturated malachite green for 5 min, water wash, safranin counterstain

- endospores are green, vegetative cell is red

- trimethoprim/pyrimethamine instead of dihydrofolate reductase

- pathway between dihydrofolate to tetrahydrofolate is blocked

- t/p is used in synergy - lower concentrations, not as competitive

* decrease *

- peptidoglycan synthesis

- metabolic/enzymatic pathway

- protein synthesis

- membrane function/destroy membrane activity/selectivity

- nucleic acid synthesis

- streptomycin

- erythromycin

- tetracycline

- initiation- GTP (energy), initiation factors, subunits (70s & 80s - E, A, P sites), start codon

- elongation- time varies, peptidyl transferase enzyme

- termination- stop codon

- structural components of the ribosome

- enzymes involved in the process

- "reading" of mRNA

- aminoglycosides

- tetracyclines

-chloramphenicol

- macrolides

- streptomycin, kanamycin, neomycin, gentamycin, tobramycin (-mycin)

- structure: amino acids and sugars: glycosidic linkage, OH, NH2 groups

- side effects: damage to 7th & 8th cranial nerves (hearing), nephrotoxicity

* concentration dependent

- blocks initiation of translation

- "locks up" the ribosome

- misreading of mRNA

- bactericidal (sulfa is static)

- can attach to 50s instead of 30s

- respiratory and electron transport system is a requirement

- not effective against anaerobes & fermenters

-  4 ring structure; different R groups

- bind reversibly to 30s ribosome near codon; block tRNA attachment

- broad spectrum antibiotic

- super infection

- shouldn't use with young children for long period due to teeth effects

- binds at 50s ribosome

- inhibits peptidyl transferase (elongation)

- broad sprectrum chemotherapeutic agent

- super infection

- Clostridium difficile- g+, intestinal flora; can cause serious colitis

- lincosamides- same action

- bind to 50s ribosome, blocks translocation

- use instead of penicillin

- outer membrane excludes the drug

- effect g+ (pneumonia)

- ex. clarithromycin, azithromycin, erythromycin

- first amino acid it formylmethionine

- initiation complex of 30s, 50s, f-met, and initiation factors

- p site- initiating tRNA carrying f-met binds here

- a site- tRNA enter to donate their amino acid; acceptor site (second codon)

- e site- tRNA exit after donating amino acid

- ribsome advances one codon (ratcheting)

- requires elongation factors

- a site becomes p site

- next codon fills a site

- sd: ribsomal binding site for mRNA, 8 pairs upstream from start codon, prokaryotes, align ribosome and start codon, anti-sd is at 16s ribosome

- k: eukaryotic verison; 3 pairs upstream

* both crucial to initiation of translation*

- due to absence of cholesterol

- polymyxin B, amphotericin B, nystatin, daptomycin

- 6 total (a-f); B & E/colistin most common; others are too toxic

- used topically (skin surface)

- hydrophobic tail, hydrophilic head

- effective against g-

- lipopeptide antibiotic

- g+ bacteria only

- cannot penetrate LPS/outer membrane of g-

- causes ionic imbalance and depolarization of the membrane (membrane permeability altered)

- anti-fungals

- polyenes- double bonds in the structure

- combines with ergosterol

- decreased toxicity

- no cholesterol

- sulfonamides

- trimethoprim

- rifampin - RNA polymerase/synthesis

- metronidazole- DNA synthesis, protozoa and anaerobes, intestines

- nalidixic acid- DNA synthesis, UTI's

- quinolones

- fluoroquinolone

- DNA is usually supercoiled for compactness, but unfolds for replication and transcription

- quinolones prevent DNA topoisomerase II/DNA gyrase, so DNA cannot unfold

- norfloxacin, ciprofloxacin, oxfloxacin (fluorine in all structures)

1. disc diffusion- zone of inhibition, kirby-bauer

2. broth dilution assays- what concentration will inhibit growth

3. antibiogram

4. broth dilution test- turbid/clear tubes in increasing concentration, MIC/MBC

5. E-strip- strip of concentrations

6. Vitek 2 system- sophisticated

MIC: minimum inhibitory concentration; want it to be low; utilize assays to find the lowest concentration of a specific antimicrobial drug needed to prevent growth; visible growth measured

MBC: minimum bactericidal concentration; lowest concentration of a specific microbial drug that kills 99.9% of cells in a given bacterial strain; assaying for live organisms in tubes with no growth from MIC

- normal flora bacteria is equally susceptible

- recall "balanced pathogenicity"

- opportunistic bacteria are part of normal flora