bisc 310 laboratory midterm 2025

protection

to keep microorganisms away from you and anything you may take out of the lab classroom

prevention

to keep microorganisms from the environment out of your work so that you can maintain the integrity of the culture you are working with

sanitizing

reduces number of microbes on a surface to a level that is considered safe

disinfecting

aims to target all microbes on a surface (viruses and bacteria)

subculturing

transferring bacteria from an old culture to fresh broth or agar

inoculation

to transfer bacteria to fresh media

four-quadrant streak designed for

allowing the isolation of single colonies of bacteria

isolated colonies often desire to provide a sample of cells that is

genetically consistent, sometimes referred to as “clonal colonies”

label plates and tube media with

your name, section number, date, organism name (or number), (and incubation temperature for plates/slants)

nutrient agar (NA)

basic lab culturing medium containing 1.5% agar that allows the medium to solidify (Nutrient Broth (NB) has same composition but w/o addition of agar

amphipathic molecules

molecules with hydrophilic polar head region and hydrophobic non-polar tail region (why soap is effective)

alcohol based sanitizers are _____ towards gram-pos and gram-neg bacteria as well as _______ towards enveloped and non-enveloped viruses, like SARS-CoV

bactericidal, viricidal

tryptic soy agar (TSA)

nutrient rich medium designed to facilitate growth of a wide range of organisms (glucose is energy source and soybean/casein provide amino acids)

fastidious

microbes with complex nutritional needs

other types of media used

sheep blood agar (SBA)

stage

platform of microscope where slide is held

mechanical stage

grip on stage where slide is held

condenser

series of lenses combined to function together below the stage

height of condenser adjusted by the

substage adjustment knob

diaphragm

controls amount of light that reaches condenser

objectives

just above the stage, attached to a rotating nosepiece, two lens systems in the microscope that are responsible for magnifying the specimen

shortest objective is aka

the low power objective (4x magnification)

longest objective is aka

the oil immersion objective (100X magnification)

40x objective is aka the

high-dry objective since it produces highest mag w/o oil

ocular or eyepiece

other lens system that provides magnification, specifically by enlarging the image that is transmitted by the objective (10x in this lab)

resolution

minimum distance between two objects which still allows them to be seen as separate (power to see two objects as separate, not same as magnification)

magnification

deals w the size of the image, extent to which an object appears bigger than true size

focus

deals with sharpness of image

numerical aperture

inherent property of the objective lense that has a fancy formula (R=λ/2NA)

never use what for microscope cleaning?

kimwipes

total magnification of the image is product of

individual magnifications provided by the objective and the ocular lenses (for example, 100x objective times 10x = 1000x magnification)

parfocal

means that once specimen is found under 10x objective, it will be in correct focal plane for other objectives as well

how to adjust ocular lenses

adjust interpupillary distance and diopter ring

calibration

process for assigning distance to the ocular micrometer

differential stains

four procedures used in prepared slides

-gram stain
-flagella stain
-capsule stain
-endospore stain

monotrichous

single flagellum

amphitrichous

has flagella at both ends

lophotrichous

tuft of flagella at one or both ends

peritrichous

has flagella surrounding the cell

capsule stains

complex stains because uses 2 stains, first positively stain with pink dye, then negatively stain with a dye that does not penetrate the capsule (physical process and not a chemical one in which a dye binds to a cellular material)

endospore stains

gram stain procedure used for these stains

flagella

specialized structure that many bacteria use for movement

motility test medium (MTM)

semi solid medium prepared as an agar deep (aka agar butt) made w or w/o indicator triphenyl tetrazolium chloride (TTC)

-as bacteria grow, ttc is reduced forming a red precipitate, allows to see if bacteria are motile (move) or not

gram+ and gram- are what kind of stain?

differential stains, also a complex stain (involves multiple reagents, unlike a simple stain)

gram+ purple

thick layer of peptidoglycan that accounts for 90% of cell wall

primary stain of gram stain

crystal violet (penetrates through both gram+ and gram- walls and binds w negatively charged bacterial components)

mordant (iodine)

2nd step of gram staining, binds to crystal violet ions to form violet-iodine clumps in the cell

ethanol

3rd gram staining step, decolorizer, removes outer membrane of gram- cells (thinner peptidoglycan becomes disrupted and crystal violet washes away)

counterstain (safranin)

4th gram staining step, no effect on gram+ bacteria but still stain decolorized gram- cells pinik

gram stain not used on ___ or ____ because they have no ______

eukaryotes, archaea, peptidoglycan

heat-fixing

adheres cells more firmly to the slide

bacterial smear aka

slide smear (start of every staining process)

shapes of bacteria in this class

coccus, rod/bacillus, spirillum, (& vibrium or corkscrews)

arrangements of bacteria in this class

diplo (2), strepto (chain), staphylo (clump)

arrangements

patterns bacterial shapes are found in

mesophiles

organisms that grow best at moderate temperatures (between 20-45 degC)

psychrophiles

aka cryophiles, grow in extreme cold (-20 to 20degC)

thermophiles

grow only at elevated temps (41 to 122degC)

optical density (OD)

aka biomass, number of bacterial cells present

(OD of 0.8 has higher bacterial density than an OD of 0.5)

canthaxanthin

red-orange pigment, part of the carotenoid pigment family

violacein

purple pigment known as violacein from chromobacterium violaceum

pyoverdine or fluorescein

yellow-green pigment, fluorescent

lag phase

first phase, organisms not dividing, adjusting to new conditions, synthesizing components needed to begin active reproduction (enzymes, cofactors, ribosomes)

exponential or log phase

rapid growth after cell division begins

generation time or doubling time

fixed characteristic for diff types of bacteria, time it takes for a bacterial culture to double i think?

stationary phase

flat line on bacterial growth curve, balanced by bacterial death

death phase

numbers of viable bacteria decrease rapidly

standard curve for bacteria

allows to estimate concentration of new OD readings

growth curve

can use standard curve to construct, shows increase and decrease in bacterial numbers over time

bacteria replicate through

binary fission (each cell splits into two)

bacterial colony shape options

punctiform, circular, rhizoid, filamentous, or irregular

bacterial colony margin options

entire, undulate, lobate, curled, filamentous

bacterial colony elevation options

flat, raised, convex, umbonate, pulvinate, crateriform

direct count of bacteria

visually count bacterial cells, time-consuming/hard

(OD) optical density

measuring absorbance of liquid culture, cannot differentiate between alive vs dead cells

viable count

measures how many live bacteria are in your culture

-make dilutions
-plate aliquots from each
-count number of colonies (countable = 30-300)

 viable count is the: No. of colonies x Plating factor x Final dilution factor

serial dilution is better than simple dilution why?

-more efficient
-uses less media/broth so there is less waste

CFU/ml

(No. of colonies x final dilution factor) / Volume of culture plated (ml)

prodigiosin

pigment, produced by Serratia marcescens only at RT/ 30℃ (water insoluble- soluble in organic solvents)

pyocyanin

pigment, produced by Pseudomonas aeruginosa (water soluble)