Micro Lab Final Study Guide

steps of gram stain

** prepare thin smear slide, air dry, then heat fix the bacteria to the slide, let cool 1. crystal violet (20 sec): will stain all cells blue/purple, rinse with DI water 2. Gram's iodine (1 min): combines with crystal violet to form insoluble complex for gram + cells aka keeps them purple, rinse with DI water 3. alcohol (5-25 sec): decolorizer. for gram positive it will dehydrate/shrink the pores = crystal violet trapped in cell wall (stays purple) - for gram neg it removes lipopolysaccharide layer so becomes colorless bc violet not retained, rinse with DI water 4. Safranin (1 min): counterstain, will color gram - cells pink / red. also sticks to gram + but can't see it due to crystal violet, rinse

Standard plate count /quantitative plating procedure

*Be able to calculate the CFU/ml in an undiluted sample, based on the statistically valid number of colonies in a set of agar plates from specific sample dilutions

Coagulase test

- All SA are coagulase positive - coagulase causes clot to form around staph infection, protecting from defenses - Clot/solids = coag positive (SA) - tube still fluid = coag neg

two main protective functions of resident microbes

1. Microbial antagonism- inhibiting one bacteria by another bacteria 2. Competition for nutrients in the ecosystem- preventing colonization of pathogens by competing for essential nutrients

understand the methods of ACP and ECP in determining microbial load of a fomite

1. Pick two fomites, and we swabbed each fomite twice (one for ACP, one for ECP) 2. Obtain 4 Falcon tubes with sterile water 3. Dip the swab in the water to moisten (a dry swab will not pick up any microbes) 4. Swab the surface of fomite #1 5. Return the swab to the Falcon tube and shake vigorously for 10 seconds in order to release the microbes from the swab into the water 6. remove the swab from the tube, throw away in biohazard 7. Lift up the top sheet of the Petrifilm, pour the contents of the Falcon tube into the center of the ACP petrifilm 8. Lay down the top sheet, and immediately place the spreader on the petrifilm 9. Gently press down until the liquid fills the spreader 10. Repeat this process for the ACP and ECP, then they need to sit for 10 minutes to solidify

tests to see if coliform is present

1. presumptive test: looking for any lactose fermenting bacteria that produce gas 2. confirmed test: determine if the bacteria is gram negative and confirms it ferments lactose 3. completed test: determine if bacteria ferments lactose and is gram neg and is non spore forming rod *required to do all 3 tests

written steps of titer

1. serial dilution 1:10, 1:100, 1:1000, 1:10,000 1;100,000 transfer mL from each tube as you go 2. obtain 5 agar tubes and take 1 ml from each dilution tube and 2 drops of E. coli and mix in agar tube 3. pour the tube into petri dishes mix, crack lid, let dry and then incubate

role of transient and residential microbes in disease transmission

Resident microbes provide some defense against disease-causing microbes and are less likely to be associated with infections, but they may cause infections in sterile body cavities, the eyes, or on non-intact skin. Transient microbes are often acquired by direct contact to a person, fomite, or environmental surface.

pure culture / purpose of it

a culture with a single kind of organism - ensures that the one bacteria we want to focus on is isolated and not contaminated by any other organism - You can rarely find a pure culture because most of the time, bacteria is in a mixed population (soil, water, and our body)

rod shape

bacilli

coliform definition and properties

bacteria that live in the digestive tract and excrements of animals and soil -gram neg, non-spore forming, ferments lactose to acid and gas -E. coli only found in feces from animals/humans

spherical

cocci -staphlococcus

arrangement: clusters

cocci in clusters staphlyococcus (staph aureus)

transient microbes

colonize on the superficial layers of the skin, are easily removed by handwashing, and usually do not multiply -do not establish themselves permanently on humans

plot optical density

concentration vs. absorbance

SPC and opitical density related

convert optical density to bacterial cells per mL and compare to SPC

PFUs

count the number of plaque-forming units *only count plates with 25-250 plaques # of plaques x dilution factor = number of phage particles in original suspension

oxidase test

determines if organism has cytochrome c oxidase in etc , if it does = aerobic respiring organism *purple = positive *clear = negative - need oxidase negative organism to do EnteroPluri test

pure culture- pour plate

dilute the bacteria to the point where they are single-celled -each cell will multiply to form a colony -colony will be identical to the progeny of the cell -dilutes one loopful of organisms with three tubes of liquified agar -advantage: requires less skill / easier -disadvantage: requires more media, tubes, and plates

how fomites are involved with disease transmission.

fomite: inanimate object that can become contaminated with pathogenic microbes and transfer pathogens to a host -person touches a fomite with a pathogen then touches their mouth/nose/eyes and the pathogen goes into their body

bacteriophage titer of a lytic bacteriophage

general: mix bacterial cells and bacteriophage in soft agar -bacteria will grow and produce lawn of growth -successful lysing = clear area on bacterial plate refered to as a plaque -each plaque is a single virus -total number of plaques = total number of virus particles in the suspension

pure culture- streak plate

goal: dilute the bacteria to the point where the bacteria are single-celled -each cell will multiply to form a colony -colony will be identical progeny of the cell -advantage: quick -disadvantage: requires skill and depends on the physical dilution of cells

the smear goals/methods

goals of smearing: -Cause cells to adhere to the slide (so they don't get washed off in staining) -ensure that shrinkage of cells doesn't occur during staining (this would cause distortion) -Prepare thin smears--thickness would prevent you from seeing individual cells/arrangements -Kill the bacteria method: - If bacteria is growing in liquid medium--place 1-2 loopfuls directly on slide - If bacteria is growing in solid media--1-2 loopfuls of water are placed on slide and then an inoculating loop is used to put organisms in the water (bacteria growing on solid media clings to each other--this helps disperse them through dilution) - If this step with water isn't done, smear will be too thick - Run the slide through a flame 2-3 times (to kill bacteria and adhere it to slide)

reading the gel

if pcr is positive - DNA has Shigella ipa gene -620 bp PCR and compare to positive control -PCR does not differentiate between live/dead cells

how infections of staph can occur

infections can occur when a break of the skin or mucous membranes (eyes, nose, mouth etc.) occurs

how do you determine the MPN

its a 3 digit number that is used to determine the number of organism/100mL 10ml samples, 1ml sample, 0.1ml sample 1. in first row of DSLB (double strength lactose broth) determine how many tubes have gas = 1st number 2. in the second row of SSLB (single strength) count how many tubes have gas = second number 3. in third row of SSLB count how many tubes have gas = 3rd number *look up 3 digit number in lab manual (code 5-2-0 = MPN of 49 organisms / 100mL and 95% confidence limits of 15 to 150 organisms)

how to convert to CFU

multiply plate count by dilution convert to 2 sig figs then get like 2.0 x 10^8 CFU/mL which is the concentration in the undiluted sample then make all the samples 10^6 then take the average

Shigella PCR assay

polymerase chain reaction -make enough copies of target DNA to analyze -using it to determine if bacteria is Shigella - positive control has 620bp band - if unknown has this band, then it is Shigella

aseptic technique

procedures used to prevent contamination of a person, object, or an area by microbes

resident microbes

reside on the surface of the skin and under the superficial cells of stratum corneum, and they have two main protective functions -commonly found in humans

our unknown enteric

salmonella - gram negative, H2S producing, anaerobic respiring bacteria. - peptone, lactose, and sucrose non-fermenter, and it ferments glucose - oxidase neg,

MSA test

selective: Presence/absence of growth differential: Agar color (ferments mannitol?) *pink to yellow = ferments mannitol (SA) *no color change = does not ferment mannitol (SS and SE)

MacConkey (MAC)

selective: inhibits G+ (bile salts and crystal violent) - growth = gram negative differential: based on the ability to ferment lactose - colors = if it ferments lactose **clear = lactose - ** Pink = lactose + (strong lactose + with pink precipitation)

Hekoen Enteric (HE)

selective: inhibits G+ (bile salts); used to recover Salmonella and Shigella differential: based on the ability to ferment lactose and produce H2S gas 3 characteristics: does it grow, what color, does it produce H2S **all green: lactose non fermenter, no H2S gas producer (shigella) *green w/ blue dots: lactose non fermenter, H2S production (salmonella) *green with bright orange: lactose fermenter, no H2S production (E.coli. / Klebsiella)

Eosin-Methylene bue agar (EMB)

selective: inhibits G+ (eosin Y & methylene blue) differential: based on ability to ferment peptone, lactose, and sucrose **amber/clear = peptone, and sucrose fermenter (NON LACTOSE) = (Salmonella, Shigella) **dark purple: peptone lactose, and sucrose fermenter (Klebsiella) **blue/black w/ metallic green: peptone, lactose, and sucrose fermenter (E.coli) - strong lactose fermenter

Why is it important to know the MIC of a disinfectant or antiseptic?

so you can know the smallest amount of disinfectant or antiseptic that will kill microorgansmism

arrangement chains

streptococci is cocci in chains (staph epidermis)

the two most common methods for testing surfaces

swabs and RODAC

SE and SS with humans

there is an increasing clinical significance, especially in immunocompromised patients -SE = most common of UTIs in hospitals -SS = second most common cause of UTIs`

confirmed test

this is the endo agar test we choose the lactose broth tube containing the most gas inside the Durham tube from presumptive test, and swab the water from that tube onto an Endo Agar plate (this is the confirmed test) Endo Agar: Selective and differential agar *Selects for gram negative bacteria to grow, and inhibits gram positive bacteria from growing *differential: can determine if bacteria ferments lactose *Has lactose in it--so it helps determine if bacteria growing can ferment lactose Conclusions: If bacteria grows on Endo Agar plate, then the organism is gram negative, and ferments lactose INTERPRETATION: *clear = lactose non fermenter *red colonies = lactose fermenter *green metallic = strong lactose fermenter

TFTC

too few to count (less than 30)

TNTC

too numerous to count (more than 300)

what are 95% confidence limits

- Need the confidence limit because we are not actually counting the bacteria, we are just making an educated guess - MPN of 49 organisms / 100mLs and the 95% confidence limit of 15 to 150 organisms. This means that the water sample had approximately 49 organisms per 100 mLs with a 95% probability of there being between 15 to 150 organisms in the water sample. provides a more accurate range of number of coliforms present in the water 95% confidence limits

Alpha toxin (Blood agar test)

- alpha toxin causes lysis of red blood cells on blood agar -presence of alpha toxin = virulence factor of SA - positive result = milky white colonies and clearing for SA - for everything else, you see growth but not clearing

Latex Agglutination Test

- rapid test for SA -Blue latex particles coated with human fibrinogen and IgG test for clumping factor or protein A (in SA cell wall) - positive = visible agglutination (clumping) of latex particles = SA -negative control = blue unsensitized latex particles that dont agglutinate

Novobiocin susceptibility test

- reveals if organism is sensitive or resistant - SS and SE are alpha neg, coag neg, ad DNAse neg -differentiates SS from SE since all other tests they are both neg -resistent = SS <= 15mm -sensitive = SE >15mm

understand concept of gram stain

- takes advantage of the dace that certain cells have distinct staining reactions - differentiates gram + and gram - based on their cell wall structure - gram + retain the purple dye, gram - are decolorized and counterstained with red dye - gram + has thick peptidoglycan, so blue dye shows up (too thick for alcohol to penetrate to make it clear) - gram - is thin peptidoglycan = dye isn't retained, so the safranin makes it pink/red

The Kirby Bauer method (antimicrobic sensitivity)

- testing sensitivity or resistance of bacteria to antimicrobials For each bacteria set up a plate: 1.Inoculate the surface of Mueller-Hinton agar using a swab to create a lawn 2.Allow 3-5 minutes for the agar surface to dry 3.Dispense disks onto surface with dispenser (6 disks testing 6 antibiotics) 4.Tap down disk with sterile loop (cooled) 5.Invert and incubate - record zones of inhibition, determine sensitive (S), intermediate (I), or resistant (R)

how resident and transient microbes may become opportunistic pathogens

- transient microbes can be opportunistic because of not washing hands after touching raw meat or a fomite or someone else. - Resident microbes can be opportunistic when you have some sort of break in the skin, someone in the hospital that is immuno-compromised.

if dilutions are outside of range

use estimated count to determine CFU/mL

EnteroPLuri test

used to ID enterics which are gram negative, oxidase negative bacteria -has 12 sections -you will get 15 results bc some sections test for more than 1 thing

standard plate count (SPC) / how to arrive at specific dilution

used to determine the number of bacteria in a sample -counts viable cells TNTC: too numerous to count TFTC: too few to count CFU: colony forming units

indicator organisms

used to determine the potential for pathogenic organisms in the water sample -E. coli

RODAC (Replicate Organism Detection and Counting)

used with smooth surfaces Specifically designed petri plates that contain a raised agar surface The agar makes direct contact with the surface and will pick up microbes like tape - Can't get in between nooks and crannies - Can be easily overloaded

bacteriophage

virus that attacks bacteria -must infect a host to reproduce

antibiotics

-Antimicrobials (usually of low molecular weight) produced by microorganisms that inhibit or kill other microorganisms -Often chemically altered to be more effective (semi-synthetic)

antimicrobials

-Compounds that kill or inhibit microorganisms -Some are chemically synthesized in the lab (not produced by microbial biosynthesis)

DNase test

-DNase agar with methyl green is a differential medium that indicates an organism's ability to produce the DNase enzyme -Coagulase positive bacteria are able to hydrolyze DNA (virulence factor) -SA produces DNase positive = dye cleared around streak line negative = no clearing

biogeochemical cycle in the winogradsky column

-Different soil microbes play key roles in biogeochemical cycling -vary based on the ability of the organism to metabolize food effectively -closed column = recycling the materials already inside -The general idea for the column is that adding various nutrients to soil will cause potential changes that will be noticeable by smell or color. Each column result will be different because everyone started with different soil

broad spectrum antibiotics

-Effective against a wider range of bacteria, typically both gram + and gram - bacteria -Helpful when the source of a bacterial infection is unknown or when the infection was caused by multiple groups

narrow-spectrum antibiotics

-Effective against either gram + or gram - bacteria -May minimize detrimental effects on gut microbiota

oxygen

-Initially, when you collect the soil and fill the column, there is oxygen present, so we would expect aerobic organisms to be present here. -Over time, the oxygen concentration at the bottom of the column decreases, becoming an anaerobic environment (decreasing O2 content as you move down the column)

nutrients

-Nutrients will affect the type of microbes growing at certain locations within the column -An egg yolk and brown paper bag pieces were added to introduce different elements to the column - nutrients in middle where they were added

light

-Some microbes may be phototrophic, so they would need light to survive, needing to be on top or on the sides of the column -Different phototrophs, such as cyanobacteria, may leave a green coloring on the side of the column (light decreases as you move inwards)

what does are the tests

-Test 5 antibiotics per species: -Broad spectrum: ampicillin, chloramphenicol (code C-30), tetracycline (code Te-30) -Narrow spectrum (Gram negative target): gentamicin (code GM-10) -Narrow spectrum (Gram positive target): vanomycin (Va-30))

brown paper bag

-carbon source -This layer may result in a brown, orange, red, or purple color in the middle of the column. The main bacteria to eat this carbon source is purple non-sulfur bacteria

ECP (E.coli/ Coliform Petrifilim)

-coliforms = red - E.coli = blue with gas production -contain indicator glucuronidase - add up red and blue dots for total coliform count

ACP (Aerobic Count Petrifilm)

-colonies indicated by red/blue dot -grows all bacteria/fungus -indicator (blue and red color) that facilitates colony enumeration - red colony = microbe growth - gives total microbial load

written out steps of typing

-create a uniform lawn of bacteria "A" and "B" on separate agar plates -incubate to dry -add 2 ul of phage

biogeochemical cycle overview

-cycling between organic (living) to inorganic (non-living) forms -organisms need nutrients to survive -mechanisms by which an organism acquires these nutrients (fermentation vs respiration) can vary -closed systems -nutrients never lost or created, just used and recycled which transports chemicals through living and nonliving parts of the planet

primers target

will target a region within the invasion plasmid antigen (ipa) group of genes -found in all 4 species of Shigella

bacteriophage typing / importance

works because each phage can only infect a single strain of bacteria -used to differentiate bacterial strains based on their susceptibility -determines the relatedness of certain bacterial species IMPORTANCE: -epidemology purposes (surveillance, outbreak investigation) ex: foodborne out break -find one bacteria from several infected people and contaminated source -isolate the bacteria like Salmonella from food source and infected people -use phage typing to determine if the isolated strain of salmonella is susceptible to the same phage - if yes, then the bacteria likely originated from the contaminated food source

Loeffler's methylene blue stain (simple stain)

-methylene blue is cationic (positively charged) dye that is attracted to negative particles like DNA/RNA in bacteria -you will see blue color when it binds to bacteria 1. add stain for 1 minute to a heat-fixed slide 2. wash with DI and blot dry -used to determine morphology and arrangement

gram positive

-no outer membrane cell wall -thick peptidoglycan layer -retains crystal violet stain (purple)

gram negative

-outer membrane cell wall (lipopolysaccharide) -thin peptidoglycan layer -does not retain crystal violet stain (PINK/RED) -more resistant to antibiotics

staph aureus infection

-particularly problematic because it can grow in salty foods and staphylococcal toxins are heat resistant. -the most clinically significant staphylococcal pathogen. -It can cause skin infections, wound infections, toxic shock syndrome, and food poisoning

lytic cycle

-phage infects a bacterium -hijacks the bacterium to make lots of phages -kills the cell by making it explode (lysis)

lysogenic cycle

-phage infects bacteria and inserts it DNA into the bacterial chromosome -allows the phage DNA to be copied and passes along with the bacterial cells own DNA

Spore Staining: Schaeffer-Fulton Method steps

-prepare the slide (add microbes then dry then heat fix) -boil water on a hot plate (fill 1/2 way with water -place paper over slide -flood paper with malachite green (all cells and spores are now green) -steam for 10 minutes -rinse with water (now cells are decolorized and spores are green) and counterstain with safranin then rinse (now cells are pink and spores are green)

gram stain for staph

-purple (gram +) -arrangement: cluster -pure: yes -morphology: cocci (irregular clusters)

how to do a serial dilution for MIC

-put 6mL of the stock solution in tube 1 -take 3 mL from tube 1 into tube 2 and repeat by taking 3mL from the previous tube into the next tube -do not add any to tube 10 = negative control, only yeast (confirms yeast is alive) -one with most concentration = tube 1 -add TSB and yeast to all tubes

TSI (triple sugar iron) slant

-selects for gram neg (inhibits gram + growth) -used to determine whether a gram neg bacteria can ferment glucose, lactose, and or sucrose **slant: tests for lactose and or sucrose ferm, butt: tests for glucose ferm -differentiates for sugar fermentation, H2S and gas production -carbohydrate fermenter = turns red to yellow -H2s production = black precipitate in butt of tube -gas production = splitting and cracking of the medium

egg

-sulfur source -darker green, purple, and / or black coloring near the bottom: - anerobic sulfate-reducing bacteria: eats sulfur, releases H2S gas

why do we test for coliforms in water samples

-to test if water is contaminated with fecal particles -coliforms are present in large concentrations, so easy to identify -the presence of E.coli can determine that pathogenic bacteria is present - tells us if water is contaminated and unsafe to drink

Optical density - determine growth by absorbance (indirect method of measuring bacteria)

-use a spectrophotometer to measure the increase in culture turbidity (measure optical density) -as cells grow, the culture becomes more turbid and a spectrophotometer can be used to measure the absorbance (optical density) -can't differentiate between living or dead bacteria

swabs

-used with irregular surfaces (used more often because it is applicable to more surfaces) -A specific area is measured and swabbed with a moistened cotton or calcium alginate swab -Swab your surface, clean your surface, re-swab the same area (before and after cleaning) -Set up ACP and ECP to look for microbial load -Disadvantage: takes more to set it up (pour on plate, incubate, read it next day)

results from entero pluri

Add each group (5 groups with 3 values each) together. This results in the 5 digit code.

completed test

After confirmed test, it is assumed that coliforms are present in water (BUT you could have non coliform bacteria present--just lactose fermenters) *So you set up a lactose broth with a durham tube and organisms from the Endo agar (to check for gas production = lactose fermentation) *Then you inoculate a nutrient agar slant from the Endo agar so you can gram stain and confirm that the bacteria is gram negative *Conclusions: In addition to being a lactose fermenter, the organism is gram neg, rod shaped, and non spore former (COLIFORM) This test is required to determine if coliforms are present in water

S. aureus virulence factors

Coagulase DNase Alpha Toxin Staphyloxanthin

column methods

Fill column half way with soil from the collection site Fill the falcon tube with water and add to column Rip paper bag into ¼-½ inch pieces and pack into falcon tube till 25mL or egg Add to column Fill with more soil until 1 inch is left at the top Fill the rest with water and seal the lid

importance of washing / reducing the microbial load on your hands

Handwashing is the single most important deterrent in transmitting microbes to each other and surfaces. Washing and reducing the microbial load on your hands protects you and the community from potential pathogens.

TSI slant: K=, A=

K= alkaline (red) A = acid (yellow)

presumptive test

Looking for any lactose fermenting bacteria that produces gas *Principle of this test: Water samples are added to tubes that have lactose broth in varying strengths If organisms present are lactose fermenters, they will metabolize *This gas is collected in a Durham tube

MPN test

MPN = most probable number = number of coliforms in 100mLs of water -uses the 15-tube method using dilution of water sample to determine the potential presence of coliforms

turbidity

Method to determine the cell number of growing microorganisms in a culture

MIC

Minimum inhibitory concentration: the lowest concentration of the chemical agent that completely inhibits growth of the bacteria (so we don't waste disinfectant) - the tube that has no growth right before the tube that has growth