BIO205L Final Exam

Safety, Ubiquity, Microscopy, Aseptic Technique, Culture Transfer, Antimicrobial Testing, Sample Stain, Gram Stain, Acid-fast Stain, Endospore Stain, Bacterial Genetics, Viral Plaques, Immunology, Media/Unknown Identification, Mac, EMB, MSA, Catalase test, Infectious Diseases, Hemolysis, Pathogenic Eukaryotes, Protozoa, Helminths, Fungi

Lab safety guidelines

PPE, wash hands/tie hair, proper disposal & report spills, use 10%/90% bleach

% bleach for bacterial spill

10%/90% bleach to water

what are fomites?

nonliving objects that transmit microbes

what is a Nosocomial Infection?

infection acquired in a hospital (HAIs)

what invert agar plates?

prevent condensation from dripping and spreading colonies

what is Ubiquity?

microbes are found everywhere (air, water, soil, surfaces, skin)

label microscope

Eye Piece (Ocular Lens)

Diopter Adjustment

Eye Piece Tube

Head

Nose Piece

Objective Lens (40x [4x objective], 100x [10x objective], 400x [40x objective], 1000x [100x oil objective])

Stage Clip

Mechanical Stage

Stage Control Knob

Aperture

Condenser

Illuminator (Light Source)

Carrying Arm

Coarse Adjustment Knob

Fine Adjustment Knob

Base

what is resolution on a microscope?

ability to distinguish two close points as separate

what is magnification?

how large the image appears

total magnification

ocular x objective

two factors that increase resolution

shorter wavelength; higher numerical aperture

why use oil at 100x?

reduces refraction → increases resolution

parfocal

stays in focus when switching objectives

simple vs compound microscope

simple = one lens; compound = multiple lenses

aseptic technique

prevents contamination of cultures, tools, and environment

inoculum

microbes being transferred

inoculation

transferring microbes to a new medium

pure culture

one species only

mixed culture

more than one species

subculture

transfer to fresh medium

turbidity

cloudiness in broth = bacterial growth

why flame loop before/after?

sterilization

why cool loop?

prevent killing bacteria

why flame tube mouth?

creates updraft → reduces contamination

antibiotics

chemical produced by microbes to inhibit other microbes

bactericidal

kills bacteria

bacteriostatic

inhibits bacterial growth

zone of inhibition

clear area where bacteria cannot grow

sensitive vs resistant

large zone = sensitive; small/no zone = resistant

factors affecting zone size

diffusion rate, drug concentration, agar depth, growth rate, drug stability

purpose of simple stain

shows shape, size, arrangement

positive stain of simple stain

basic dye binds to cell

negative stain of simple stain

acidic dye stain background; no heat fixing

why thin smear? of simple stain

prevents clumping; easier to see cells

why air dry? of simple stain

prevents boiling during heat fixing

why heat fix? of simple stain

kills cells + adheres them to slide

two reasons for negative stain - of simple stain

no distortion; good for capsules

capsule function

prevents phagocytosis; increases virulence

can negative stain show internal structures

no

coffee stain question

coffee is not a biological stain; no ionic binding

positive stains of simple stain

crystal violet, methylene blue, safranin, malachite green

negative stains of simple stain

nigrosin, india ink

primary stain of gram stain

crystal violet

mordant of gram stain

iodine

decolorizer of gram stain

alcohol

counterstain of gram stain

safranin

gram+ cell wall of gram stain

thick peptidoglycan

gram- cell wall of gram stain

thin peptidoglycan + outer membrane

most crucial step of gram stain

decolorization

forget safranin of gram stain

gram- appear colorless

use methylene blue instead of safranin of gram stain

gram- appear blue

no mordant of gram stain

gram+ may wash out

over-decolorize of gram stain

gram+ look gram-

under-decolorize of gram stain

gram- look gram+

gram variability of gram stain

mixed purple/pink staining

causes of gram+ appearing gram- of gram stain

old culture, over-decolorization, damaged cell wall

medical importance of gram stain

guides antibiotic choice

harder to treat: gram+ or gram-?

gram- (outer membrane)

urine left out over weekend

autolysis → gram variability

primary stain of acid-fast stain

carbol fuchsin + heat

decolorizer of acid-fast stain

acid-alcohol

counterstain of acid-fast stain

methylene blue

acid-fast cell wall of acid-fast stain

mycolic acid

why heat? of acid-fast stain

drives stain through waxy wall

diagnostic value of acid-fast stain

identifies TB and leprosy

two acid-fast bacteria of acid-fast stain

M tuberculosis; M. leprae

unqiue molecule of acid-fast stain

mycolic acid → chemical resistance

primary stain of endospore stain

malachite green

counterstain of endospore stain

safranin

spore color of endospore stain

green

vegetative cell color of endospore stain

red

purpose of steaming of endospore stain

forces dye into spores

is sporulation reproctive?

no - survival mechanism

two spore-forming bacteria

bacillus anthracis; clostridium tetani

chemical in spore coat

dipicolinic acid

competency

ability of cells to take up DNA

transformation experiment

pGLO plasmid → GFP + ampicillin resistance

plasmid characteristics

circular, extrachromosomal, self-replicating DNA

PCR components

templete DNA, primers, nucleotides, Taq polymerase, buffer

PCR steps

denaturation → annealing → extension

how PCR amplifies DNA

each cycle doubles target DNA

agarose gel electrophoresis

DNA moves toward positive electrode; smaller fragments move faster

why incubate virus with bacteria first?

viruses need host cells to replicate

what happens at higher dilutions?

fewer plaques

plaque formation

virus infects → lyses cells → clear zone

PFU formula

PFU/mL = plaques divided by (dilution x volume plated)

humoral immunity

B cells; antibodies

cellular immunity

T cells; cytotoxic killing

antigen

molecule that trigger immune response

antibody

protein that binds antigen

immunoglobulin

antibody class

ELISA purpose

detects antigen/antibody; track disease outbreaks

Selective medium

allows some bacteria to grow; inhibits others

differential medium

all grows; show differences

MAC selects for

Gram- enterics

MAC differentiates by

lactose fermentation

MAC result fro E. coli

pink colonies (lactose+)

MAC result for non-lactose fermenters

colorless

EMB selects for

Gram-

EMB differentiates by

lactose fermentation