MBI 161 Lab Exam

Fermentation

Common microbial catabolic pathway that begin with sugar and ADP and produce ATP with acids or alcohols

Beta-hemolysis

Complete hemolysis, clear zone on blood agar, indicates Streptococcus pyogenes that produces hemolysins (can cause streptococcal pharyngitis, impetigo, cellulitis, or necrotizing fasciitis)

If no treatment is applied what does a lawn of bacteria look like?

Completely covers agar in plate; creates a complete spread of growth

Acid-fast stain

Complex and differential

Endospore stain

Complex and differential

Gram stain

Complex and differential

What does this media detect? How?

Detects lactose-fermenting organisms by

Which media type allows visual grouping of bacteria by metabolic characteristics but does not affect growth?

Differential media

Why and how is an endospore stain differential?

Differentiates between endospores and vegetative cells by targeting endospores' protective layers (resist staining)

Be able to calculation dilution factors and number of viable cells/mL in an original culture

Dilution factor: (mL transferred to tube / (mL transferred + mL already present)) x factor for undiluted culture Viable cells: (#colonies/mL plated) x (1/dilution factor)

How do you perform a spread plate?

Dip sterile swab into culture --> rub swab over entire NA surface --> turn plate 90 degrees --> rub swab over entire NA surface again

Differential medium

Formulated to allow one type of bacteria to be distinguished from all or most other types, generally via color change of medium or colony due to metabolic processes of the bacteria

Function/composition of selective media (S)

Formulated to inhibit growth of certain bacteria types while allowing others to grow

Selective medium

Formulated to inhibit growth of certain types of bacteria but allow others to grow; useful for bacteria identification

Pipet

Glass or plastic cylinder used for transferring liquids; have markings so precisely measured amounts can be transferred; used with pipet aid

What color are Gram+ and Gram- cells after each step of a Gram stain and why?

Gram+ stains purple Gram- stains red

What are the characteristics of fecal coliforms?

Gram-, lactose fermenting, enteric bacteria

Selective-differential media

Has characteristics of both selective and differential medium; useful for bacteria identification

Simple stain

Involves use of just one dye; can be used to give color to bacteria and other cells so they are more easily visualized

Complex stain

Involves use of more than one dye

How do you test penetrating ability of UV?

Keep one plate unexposed to UV light as control; cover one plate with bibulous paper and another with the petri dish lid and expose to UV light for 3 minutes

Hemolysins

Membrane-disrupting exotoxins that disrupt/lyse RBCs (hemolysis)

YNB agar

Minimal medium, no growth factors such as amino acids

Culture medium

Mixture of nutrient materials used to grow bacteria; solid form of agar in petri dish or test tube (called slants), liquid form of broth in test tube

Be able to read/interpret salt plates results: what would growth pattern be for most bacteria versus halotolerant?

Most bacteria could not grow with salt present, or past 5% NaCl; halotolerant can show growth well past this threshold

Be able to calculate phage/mL in original (undiluted) phage solution

(#plaques/mL plated*) x (1/dilution factor) *mL of E coli or mL of bacteriophage?

Be able to read and interpret results for presumptive test

Any gas produced (bubble present in broth) indicated presence of lactose-fermenting bacteria; acid production indicated by yellow in medium

Which genera are endospore formers?

Bacillus spp. and Clostridium spp.

Negative stain

Background of slide is stained, leaving clear areas where structure of interest can be seen

How can a mutant in an amino acid synthesis pathway be recognized?

Bacteria does not grow on minimal agar (such as YNB agar)

Culture

Bacteria multiplying in or on culture media; culture growing on solid media will form colonies on surface; culture growing in liquid media appears as cloudiness in broth, aka turbidity, amount corresponds with amount of growth; broth cultures must be mixed before use because bacteria settle at bottom of tube

Starter culture

Bacteria, molds, yeast; known microorganisms used to start fermentation reaction

Substrate

Beginning materials

Gamma-hemolysis

No hemolysis, no change on blood agar

Isolated colony

Not visibly touching any other colony; can be used to create pure culture

Alpha-hemolysis

Partial hemolysis, greenish zone on blood agar

Enterotubes

Read results/identify unknown organism using tube if provided with tables

Product

Resulting materials

Neopeptane agar

Rich with amino acids

Spirillum

Rigid helix structure

Bacillus

Rod shaped

Coccus

Round/spherical

Composition, media type (S/D/S-D), how media works, which exoenzyme/metabolic activity media detects and what are their activities, how to read/interpret results: MacConkey's agar

S (contains bile salts/crystal violet to inhibit Gram+ non-enteric bateria) and D (contains lactose and pH indicator dye); appearance of reddish/pink color in bacteria colonies and surrounding media indicated ability of bacteria to ferment lactose

Differential stain

Takes advantage of structural differences among bacterial species that cause reactions to dyes to differ; allow bacteria to be sorted into groups which is important when trying to identify them

Why and how is an acid-fast stain differential?

Targets cells with mycobacterial cell walls

Why and how is a Gram stain differential?

Targets structural differences in bacterial cell walls (peptidoglycan)

Why is a 3-phase streak performed?

To obtain isolated colonies

What disease can be diagnosed using an acid-fast stain?

Tuberculosis (TB)

Be able to read/interpret results of genetic diversity

Used 12 isolated colonies to create 12 pure cultures on one place of NA and one of YNB agar; pick one that did not grow on YNB and create lawn on another plate with amino acids; if it grows around amino acid disc, mutant bacteria cannot synthesize that amino acid on its own

Incubator

Used to grow bacteria at specific temperature; petri dishes placed upside down to keep condensation from forming on lid and dripping onto agar sample

What colors are vegetative cells and endospores on an endospore stain?

Vegetative cells stain red Endospores stain green

Bacteriophages

Viruses that infect bacteria

Colony

Visible group of bacteria that arises on solid culture medium from single bacterium, single endospore, or group of bacteria that grow in clusters/chains; bacteria in colony are exact copies of original cell that was placed on medium

What does the presence of fecal coliforms indicate about the water sample (confirmative)?

Water is not safe to drink because fecal coliforms indicate fecal contamination and possible presence of pathogens

Parfocal

Ability of microscope to maintain some focus when switching between objectives

Magnification

Ability to enlarge objects

Resolution

Ability to show fine detail and distinguish 2 adjacent objects from one another

Why would you heat fix a slide?

Allows sample to take in stain more easily, helps bacteria stick to slide

With what objective lens should the coarse and fine adjustment knobs be used?

Coarse can be used with 4x and 10x, fine should be used with 40x and 100x

Types of cultures

Pure, mixed, contaminated

Lawn of growth

Colonies growing on the surface of a solid culture media close enough that individual colonies are indistinguishable

What bacteria are considered "acid-fast" and "non-acid-fast"?

Acid-fast bacteria have mycobacterial cell walls (ex. M. tuberculosis), non-acid-fast do not have mycobacterial cell walls

What colors are acid-fast and non-acid-fast bacteria on an acid-fast stain?

Acid-fast stain red Non-acid-fast stain blue

How do you perform a 1/10 dilution?

Add 1.0 mL of bacterial broth to 9.0 mL sterile water

When and why should heat be applied to an acid-fast stain (not including heat fixing slide)?

After applying carbolfuchsin to drive stain into mycolic acid

When and why should heat be applied to an endospore stain (not including heat fixing slide)?

After applying malachite green to drive stain into endospores

How do plaques form?

1 bacteriophage --> infects one bacterial cell --> cell lysis --> release of many phage --> infect surrounding cells --> cell lysis --> plaques

What is the equation for the yeast fermentation of sugar (including all substrates and products)?

1 glucose + 2 ADP --> many reactions --> 2 ethanol + 2 CO2 + 2 ATP

Know how to perform a 1/10 dilution, series of dilutions, and how to plate a dilution

1/10: 1 mL sample into 9 mL saline Series: repeat 1/10 dilution Plate: use pipet to transfer specific measurement

If you transfer 1 mL from a 10^-5 dilution to a tube containing 9 mL of sterile saline, what is the final dilution factor?

10^-6

If you count 45 isolated colonies on a NA plate, which was inoculated with 0.1 mL of a 10^-5 dilution of an original culture, how many viable cells/mL were in the original culture? (Show work and express answer in scientific notation w/units)

=(45/0.1 mL) x (1/10^-5) =450 x 10^5 =4.5 x 10^2 x 10^5 =4.5 x 10^7

Intermediate

???

Function/composition of selective-differential media (S-D)

Both selective and differential components

What reagents are used in an acid-fast stain in order and what are their functions?

Carbolfuchsin, primary stain --> acid alcohol, destain --> methylene blue, counterstain

How do you prepare a smear?

Catch drop of water with inoculating loop --> apply to clean slide --> aseptically transfer bacteria/fungi from NA to water droplet --> mix cells in water with loop --> allow samples to dry completely --> heat fix

What is the structural difference between Gram+ and Gram- cell walls that the Gram stain is based on?

Cells with thick peptidoglycan stain Gram+, cells with thin peptidoglycan stain Gram-

Plaques

Circular clearings in a lawn of bacteria

How would capsule producing bacteria appear after this stain is performed (how would it look on the slide)?

Clear zone visible around cells

All formulations of culture media must...

Contain source of energy (ex. glucose), carbon, nitrogen, sulfur, phosphorus, any other compounds required by species, sufficient moisture (water), proper salt concentration, be at proper pH, be sterile before use; solid forms must contain agar

Mixed culture

Contains 2 or more known species or microorganisms

Contaminated culture

Contains both known and unknown (unwanted) microorganisms

Pure culture

Contains only one known species or type of microorganism

How do you test the time to kill using UV?

Cover half of three plates with cardboard and leave the other half uncovered; expose one to UV light for 30 second, one for 1 minute, and one for 3 minutes

How can the specific pathway disrupted be identified?

Creating a culture on another plate to test if bacteria can form when amino acid is present

What reagents are used in a Gram stain in order and what are their functions?

Crystal violet, primary stain --> Gram's iodine, mordant --> alcohol, destain --> safranin, counterstain

Vibrio

Curved rods

Composition, media type (S/D/S-D), how media works, which exoenzyme/metabolic activity media detects and what are their activities, how to read/interpret results: Skim milk agar

D; contains milk protein (casein) which makes media appear cloudy; clearing of opacity around growth indicates ability to produce/secrete proteases (enzymes that digest casein) and bring casein into cell, resulting in loss of cloudiness

Composition, media type (S/D/S-D), how media works, which exoenzyme/metabolic activity media detects and what are their activities, how to read/interpret results: Starch agar

D; contains starch which can be seen after growth by covering surface of plate with iodine (Gram's iodine) solution, turns brown/purple when reacting with starch; lack of color formation after applying iodine indicates production/secretion of amylases to break down starch, thus no color develops

Composition, media type (S/D/S-D), how media works, which exoenzyme/metabolic activity media detects and what are their activities, how to read/interpret results: Spirit blue agar

D; large lipids complexed to blue dye; loss of blue color in area around bacterial growth or appearance of blue colonies indicates ability to produce/secrete lipase to digest lipids outside cell and bring into cell, causing loss of color

Composition, media type (S/D/S-D), how media works, which exoenzyme/metabolic activity media detects and what are their activities, how to read/interpret results: DNase agar

D; large molecules complexed to green dye; loss of green color indicates ability to produce/secrete DNase to breakdown DNA molecules and bring them into cell, resulting in loss of color

How does spontaneous mutation occur?

DNA polymerase makes errors during DNA replication

Why is a capsule stain a differential stain?

Distinguishes cells with glycocalyx capsule

ATP

Energy carrier for cells used when energy is required for biosynthetic pathways

What are exoenzymes?

Enzyme that acts outside of the cell that produces it

What media is used for the confirmed test for fecal coliforms?

Eosin methylene blue (EMB) agar

What important fecal coliform does this media detect? How?

Escherichia coli (also Enterobacter spp.); media prevents Gram+ cells from growing, dyes change color with amount of acid produced (E. coli appears bluish-black with metallic green shen, Enterobacter spp. appear deep pink/purple and mucoid/glossy)

Which products during the fermentation exercise were detectable and how were they detected using the fermentation apparatus?

Ethanol - yeasty smell CO2 - bubbles form in the vertical tube of apparatus

Why is a capsule stain a negative stain?

Everything stained but capsules

What does a positive test indicate about the bacteria present in the water sample (presumptive)?

Fecal coliforms may be present

Spirochete

Flexible spring spiral

Be able to read and interpret results of disc method

Identify zone of inhibition in lawn; no zone is resistant, zone present is susceptible

Oil immersion

Immersion oil used with 100x objective to reduce light refraction to ensure enough light enters objective lens

Be able to identify plaques on a bacterial lawn

Kk

Be able to read and interpret results from confirmed fecal coliform test

Kk

Be able to read and interpret the results of UV penetrating ability and time

Kk

Know how to perform a 3-phase streak.

Kk

Look over parts of the miscroscope

Kk

What media is used for the presumptive fecal coliform test?

Lactose fermentation broth

Inoculating loop

Looped wire attached to a handle used to transfer bacteria; must be sterilized (incineration)

Total magnification

Magnification of ocular lense (10x) x magnification of objective (4x, 10x, 40x, 100x)

What reagents are used in an endospore stain in order and what are their functions?

Malachite green, primary stain --> water, destain --> safranin, counterstain

Know how to perform disc method experiment

Place disc containing agent onto spread plate of bacteria, incubate to allow bacterial lawn to grow

What does the presence of fecal coliforms indicate?

Presence of fecal contamination or sewage

Purpose of genetic diversity lab

Screening for mutants

Mannitol salt agar (MSA) test

Selective (high salts selects for halotolerant) and differential (only Staph. areus ferments mannitol to produce acid so pH indicator turns yellow in media, no color change with Staph. epidermidis and others)

Which media type affects growth?

Selective media

Know how to perform bacteriophage plaque assay

Serial dilution of bacteriophage then plate diluted phage with excess bacteria

Function/composition of differential media (D)

Should allow all bacteria types to grow, formulated to allow one type of bacteria to be distinguished from all or most other types usually by chemicals that change colors under certain conditions

Capsule stain

Simple, negative, differential

Inoculum

Small amount of bacteria that are transferred to sterile medium and multiply to form culture; performed using an inoculating loop or pipet

Which important skin normal microbiota and skin pathogens are halotolerant?

Staphylococcus epidermidis and Staphylococcus aureus

Is UV used for sterilization, disinfection, and/or antiseptics? Why?

Sterilization (kills all metabolically active cells) and disinfection (eventually can kill all endospores; cannot be used for antiseptics because it would kill living tissue