Learn: Microbiology Lab Midterm (Labs 1-4) Auburn

these 3 things are personal protection equipment (PPE) requirements

wear closed toed shoes, gloves and lab coat while in the lab

what disinfectant is used in the lab?

lysol

where are items disposed of that have been in contact with bacteria? (ex: petri plates, gloves etc.)

biohazard can

where are slides, swabs and pipettes disposed of?

sharps container

glassware

discard cart

where do items that are not contaminated with bacteria (gum, paper products, items brought into laboratory) go to be disposed?

regular garbage can

these are microorganisms not known to cause disease in healthy adults

BSL 1

these are indigenous microorganisms that can lead to diseases of varying severity in healthy adults. (ex: Staphylococcus aureus, Streptococcus pyogenes, and Salmonella)

BSL 2

these are indigenous or exotic microorganisms that cause serious or potentially lethal disease through respiratory transmission (ex: Mycobacterium tuberculosis, Bacillus anthracis, Francisella tularensis)

BSL 3

these are microorganisms that are dangerous and exotic with a high risk or aerosol transmitted infections. Rarely are there treatments or vaccines for these microorganisms, and the diseases they cause are frequently fatal (ex: Ebola virus, Marburg virus)

BSL 4

this is a general term for living organisms that cannot be seen without a microscope, even though some people include viruses and porins in this group

microorganisms

this refers to the concept that microorganisms are everywhere (air, food, water) and they are only not found where they are intentionally excluded

ubiquity of microorganisms

practicing aseptic technique prevents contaminating these 3 things:

yourself, others, and the media/ microorganisms

these destroy vegetative cells and viruses but may not destroy endospores

disinfectants

these are used to transfer cultures of bacteria

loops and needles

this is a sterilization technique where you place your utensils into the flame of the bunsen burner until it is red hot

flaming the loop

how long should you wait for your utensil to cool after flaming?

15-20 seconds

how do you aseptically remove the cap from a test tube?

using the pinky finger of your dominant hand

how do you sterilize a test tube?

flaming the mouth of the tube before and after you inoculate it with bacteria

what is the correct orientation for a petri plate?

lid down, bottom up

where and when was the first microscope developed?

Middleburg, Holland in 1590

this microscope is the simplest form of microscopy where light is either passed through, or reflected off a specimen. Illumination is not altered by devices that change the properties of light (such as polarizers or filters)-- requires the use of stains to visualize cells

bright field

this microscope converts the differences in optical density (refractive index) of cells into different shades of brightness (contrast). It allows for the visualization of morphology, external structures and some internal structures-- stains are not required

phase contrast

this microscope's contrast is created by a bright specimen on a dark background. it is ideal for revealing morphology and external structures, but it does not provide a great deal of information about internal structures-- stain is not required

dark field

this microscope uses high intensity illumination to excite fluorescent molecules (fluorophores) in the sample. when a molecule absorbs photons, electrons are excited to a higher energy level. as electrons 'relax' back to their ground-state, vibrational energy is lost and as a result, the emission spectrum is shifted to longer wavelengths. fluorescence emanates from the sample-- requires the use of fluorophores

fluorescent

this type of microscope will be used in lab

bright field

on the brightfield microscope, this is the horizontal area that supports the microscope slide

stage

the framework of the brightfield microscope includes

arm and base

the brightfield microscope's light source is

LED

the brightfield microscope has a 3 lens system that includes

oculars, objectives and condenser

the ocular lenses have _____ magnification

10X

the objective lenses have ____________ magnification

10X, 40X and 100X

this is determined by multiplying the ocular power by the objective power

total magnification

the ________ collects and directs the light from the light source to the slide and is located under the stage-- does not affect the magnification power

condenser

the amount of light that reaches the slide is controlled by a _______ located inside the condenser

diaphragm

immersion oil has the same refractive index as ________

glass

this fills the gap between the objective and slide to form a continuous lens path which increases image resolution by reducing light refraction

immersion oil

this is the ability of a microscope to remain relatively in focus when changing from a lower power objective to a higher power objective

parfocal

this is a mathematical expression that describes how the condenser lens concentrates and focuses the light rays from the light source

numerical aperture

this is the ability of a lens system to show closely spaced objects as distinct and separate

resolving power

this is the distance between the bottom of the objective lens and the slide

working distance

why are bacteria transparent when viewed by the brightfield microscope?

they are composed of 80% water

this is used as a simple stain

methylene blue

the most common mistake when performing a smear prep is

placing too much bacteria on the slide

this type of stain is useful in determining cell morphology, cell arrangement, and internal storage materials

simple stain

these have spherical morphology and may appear singularly, in pairs (Neisseria), in groups of four (Micrococcus), in chains (Streptococcus) or in clusters (Staphylococcus)

cocci

these have rod shaped morphology and may occur singularly (Psuedomonas aeruginosa) or in chains (Bacillus)

rods

these have corkscrew morphology (Campylobacter)

spiral/ curved

who created gram staining and why did he do it?

Hans Christian Gram was attempting a staining technique to differentiate bacteria from eukaryotic nuclei of lung tissue

this bacteria retains crystal violet stain because of its thick peptidoglycan layer

gram positive

this bacteria does not retain crystal violet because it has a thin peptidoglycan layer

gram negative

this is the counter stain used when performing gram staining technique

safranin

this is the primary stain used when performing gram staining technique

crystal violet

the difference in stain uptake due to cell wall composition makes the gram stain a _______

differential stain

steps of gram stain

crystal violet, iodine, ethanol, safranin

what will happen if the slide is heated for too long?

the glass will break and the organisms membrane will rupture

what is the purpose of Gram's iodine?

it plugs the pores in gram positive cell walls, so the ethanol will not wash out the crystal violet

what color does gram positive bacteria stain?

purple

what color does gram negative bacteria stain?

pink/red

this is a medium that will allow only certain microorganisms to grow and will inhibit the growth or other microorganisms-- anitbiotics, antifungals, dyes and inhibitory compounds are included to give it these properties

selective medium

the incorporation of sodium chloride (salt) in the mannitol salt agar (MSA) selects for the genus ________ but inhibits the growth of bacteria that cannot tolerate the salt concentration

staphylococcus

this is a medium that differentiates microorganisms based on their appearance-- contains substances that cause some bacteria to take on appearance that distinguishes it from other bacteria

differential medium

when this bacteria grows on MSA, it ferments mannitol producing acid and changing a pH indicator in the media from red to yellow around the colonies

Staphylococcus aureus

this is an example of a differential media where some species of Streptococcus and Enterococcus produce exotoxins (poisons) that completely lyse RBC in the agar

sheep blood agar

complete lysis of RBC around a colony is known as _______ and results in a clear zone around the colonies

beta hemolysis

other species partially breakdown RBC producing a greenish discoloration around the colonies known as

alpha hemolysis

species of bacteria that do not exhibit any hemolysis of blood display ________ and have no effect on the RBC in the agar plate

gamma hemolysis

when aerobic bacteria grow by respiration, this chemical is a by-product of the reaction

hydrogen peroxide (H2O2)

aerobes produce the enzyme ________ which degrades damaging hydrogen peroxide into harmless oxygen and water

catalase

this is bacteria that cannot grow in the presence of oxygen

strict anaerobes

this is bacteria that are considered anaerobes but can tolerate oxygen

aerotolerant

the presence of the enzyme _______ is one way to differentiate these bacteria (anaerobes) from aerobes or facultative anaerobes

catalase

this is bacteria that can grow by respiration or fermentation with or without oxygen

facultative anaerobes

if an organism can breakdown carbohydrates, this is usually produced

acid

the presence of _____ is indicated by a color change in the pH indicator phenol red-- from red at basic pH to yellow at acidic pH

acid

T/F: any carbohydrate can be added to the phenol red tube

true

what type of media is the phenol red tube?

differential

starch macromolecule consists of two constituents such as

amylose and amylopectin

this is a straight chain polymer consisting of 200-300 glucose molecules

amylose

this is a larger branched polymer of glucose

amylopectin

bacteria that hydrolyze starch produce ________ that degrade the starch molecule into molecules of maltose, glucose and dextrins that can enter the bacterial cell

amylase

starch hydrolysis is detected by adding _______ to a starch agar plate

gram's iodine

the starch agar plate is what kind of medium?

differential

iodine complexes with the starch macromolecule causes the starch agar to turn black/ brown; however if the starch in the medium has been degraded by ______ produced by the bacteria, the starch agar adjacent to the bacterial growth will be clear

amylase

bile esculin agar is what kind of medium?

selective and differential medium

the bile esculin agar is used to identify these bacteria; it contains oxgall (bile salts) which inhibits the growth of other gram positive bacteria

Enterococcus and group D Streptococcus

when an organism hydrolyzes esculin to form esculetin and dextrose, the esculetin reacts with ______ to produce a dark brown/ black medium

ferric citrate

this test is an excellent definitive test for identifying Staphylococcus aureus

coagulase test

in the coagulase test, you inoculate a tube of _______ with bacteria, and if it clots, the organism produces coagulase and is considered positive.

blood plasma

______ can give a false-positive reaction to the coagulase test

Pseudomonas

in the catalase test, what indicates a positive result?

bubbles

in the bile esculin test, what indicates a positive result?

blackening of the tube

in the bile esculin test, what indicates a negative result?

no color change or less than 1/2 of the slant is black

in the catalase test, what indicates a negative result?

no bubbles

in the coagulase test, what indicates a positive result?

clot formation

in the coagulase test, what indicates a negative result?

no clot formation

in the MSA test, what indicates a growth +/ acid+ result?

yellow colonies from acid produced by mannitol fermentation

in the MSA test, what indicates a growth +/ acid - or growth -/ acid - result?

colorless colonies or no growth

in the phenol red tubes test, what indicates a positive result?

yellow color

in the phenol red tubes test, what indicates a negative result?

red color