OSU Microbiology 4000: Lab Practical. Everything.

Autotrophic

produce own food source from inorganic carbon and energy sources.

Bacteriostatic

Antibiotic that inhibits bacterial growth.

Precipitin Test steps

(BSA) Antibody in 1 capillary tube, then draw up antigen from another capillary tube then place in clay and see what precipitates.

Trypticase Soy Agar

(TSA Plate) general plate with many nutrients. innoculate at 30 deg C. Same temp as body, helps with growth

prokaryotes

(do not have visible cilia and flagella under a light microscope) Cells that do not contain nuclei

eukaryotes

(have visible cilia and flagella under a light microscope) Cells with internal membranes

Constructive milk plated

Buttermilk

alkaliphile

microbes that show optimal growth at high pH

Current flow

where the movement of all objects are in one direction due to a current

Brownian motion

where the objects bounce around in all random directions and vibrate around

petri dishes, used gloves, contaminated paper towels

white biohazard container

Can E. coli survive on MM1? Z. salivarius? S. epidermis?

yes, no, no

Objective's we used in lab

40, 10, 100

nonhalophiles can grow in

0-2%

Describe the KIA (6)

1 red slant and a yellow but is glucose fermentation, 2 lactose and glucose fermentation is yellow slant and yellow but 3 protein utilization is a red slant and no change in butt (staying orange) or red throughout. 4 Gas is produced in the yellow slant, yellow butt, cracking the agar. 5 in red slant yellow but a black precipitate can form signifying desulfhydrase activity creating FeS. 6. remaining orange is nothing utilized.

Magnification of Compound bright field microscope

10 (ocular magnification) x ? (objective magnification)

Total magnification we used in lab

100, 400, 1000

How many times do you use buffer in ELISA

3 steps involve washing with wash buffer

What was the best antiseptic?

3.0% H2O2

Size of paramecium

50-300 micrometer eukaryote, protazoa

endospore

A thick-walled protective spore that forms inside a bacterial cell and resists harsh conditions. (Bacillus and Clostridium, undergone sporulation, therefore can survive even prolonged exposure to moist heat at relatively high temperature)

Differential Acidic dyes

Acid fuchsin

Bactericidal

Antibiotic that can kill bacteria.

Agglutination reaction

Ag-Ab complexes form when antibodies react with insoluble antigens, which are located on invading cell surfaces. This reaction causes the cells to lump together, making it easier to remove the invaders from the body by phagocytosis.

Preciptin reactions

Ag-Ab complexes form when antibodies react with soluble antigens; the insoluble complexes precipitate from solution and are removed from the body either by phagocytosis or filtration.

Differential media

Allow a variety of organisms to grow, but enable the observer to visually distinguish among different types of bacteria based on how they metabolize the components of a given medium.

Thymine dimers

Alter the genetic code of a cell affecting replication and transcription, resulting in cell death.

Osmotic pressure

Amount of pressure needed to stop the net flow of water across a semipermeable membrane. Most bacterial habitats are hypotonic.

The kirby-bauer method

Antibiotics dispensed on plate, if zone of inhibition is there then the zone of inhibition is inoculated onto another plate to determine if something is bacteriocidal, or bacteriostatic.

Disinfectants

Applied topically to inanimate materials.

in the precipitin test, the soluble antigen is

BSA

Symbiotic relationship of yogurt

Bacteria make the yogurt more acidic.

microaerophiles

Bacteria that use oxygen, but only at a low concentration

Transient microbiota

Bacteria, mold spores, etc. that we have on our skin surfaces from touching objects in our environment.

What do anti rhesus antibodies agglutinate?

Blood with rhesus antigen

What do anti-a antibodies agglutinate?

Blood with the A antigen.

What do anti-b antibodies agglutinate

Blood with the B antigen

Citrate

Blue=positive, means pH change and the ability of the organism to use citrate as a carbon source.

Stomacher

Breaks up a sample using the same type of physical action that the human stomach produces.

Units for TVC?

CFU/mL

facultative anaerobes

Can make enough ATP to survive using using fermentation or respiration. (with or without oxygen)

Acid-fast bacteria

Can't be stained under normal circumstances due to mycolic acid. Technique involves primary stain, two mordants, decolorizing agent, and counterstain.

obligate anaerobes

Cannot survive in the presence of oxygen

Describe phenol red carb. broth test

Carbohydrates have been used if the solution turns yellow, it is red only nothing was used, red and cloudy then peptone was used, yellow with a little air bubble in the durham tube is gas produced and carb. used.

Plasmolysis

Cell membrane is pulled away front he cell wall, causing the bacteria to die.

IMViC

Collectively the MRVP, citrate, and indole tests

How did we test the effectiveness of ampicillin and kanamycin on transformation

Competent cells- transfer 50 microliters in plasmid A, 50 microliters into p B. - Ice for 20 minutes those two, heat shock 42 Celsius for 90 seconds, then place in ice for 2 minutes. -Plate plasmid A on half of the ampicilin plate, half of the kanamycin plate then do the same for plasmid B on the opposite side of each.

What does the UV radiation do to the bacteria to control them

Creates the thymine dimers and damages the DNA backbone.

Simple stain Basic dyes

Crystal violet, methylene blue

Differential stain Basic dyes

Crystal violet, safranin, malachite green

Bacteria that isn't stained would be viewed using

Darkfield or phase contrast.

What are examples of gram -, + and fungi in biofilm

E. coli, s. aureus, Candida Albicans.

are Catalase and Cytochrome oxidase endo or exoenzymes

Endoenzymes

Virulence factors

Enhances ability to evade host defenses and cause disease. -Capsule -Enzymes -DNase -Coagulase -Hemolysins

Nitrate reduction

Enteric facultative anaerobes in the absence of oxygen use nitrate as a terminal electron acceptor resulting in reduction of nitrate (NO3) to nitrite (NO2) or ammonia (NH3.)

ELISA

Enzyme-Linked Immunosorbent Assay. Employs the use of secondary antibodies that are conjugated to a specific detector enzyme.

Peptidases

Enzymes produced by bacteria to break down proteins into small peptides or amino acids. Some are exported from the cell into the environment where they become catalytically active.

Budding

Form of asexual reproduction used by yeast cells.

MacConkey Agar Plate

Gram-negative enterics usually grow on these plates. If the colonies are hot-pink the organism can ferment lactose and are coliforms. If the colonies are light yellowish, the organism can't ferment lactose.

Extreme thermophile

Growth at 80C or higher

Psychrotroph

Growth between 20-30C

Mesophile

Growth between 25-40C

Thermophile

Growth between 45-60C

What is hypotonic, hypertonic and isotonic

Hypertonic is a lot of solute in the environment drawing the water out. Hypo is the opposite.

Starch-agar plate

If starch has been utilized, there will be a clear area around the streak, indicating that the organism can hydrolyze starch and is alpha-amylase positive.

How are nitrogen reduction and denitrification identified in the nitrate reduction procedure?

If the tube doesn't change color after the sulfanilic acid and naphthylamine, and the zinc dust and there is a bubble in the durham tube-> denitrification. If there is no bubble -> Nitrogen reduction. Red color after the first test -> NO3- -> NO2- stop

Antibiotic modes of action (4)

Interfere with cell wall synthesis Inhibit protein synthesis Inhibit nucleic acid synthesis Negatively alter plasma membrane

Endoenzymes

Intracellular peptidases, catalytically active inside the cell.

Coarse focus knob

Larger knob on inside used to rapidly change the focus

Compound bright-field microscope

Light microscopes, specimens appear dark against bright background. "Bright field." (the most commonly used)

dark field microscope

Light source is above specimen, creating dark background and light image

What was the best disinfectant?

Lysol

Describe MR test

Methyl red is a red pH indicateor used in examining the amount of acids due to glucose fermentation. If the amount of acid is low the indicator remains yellow or stays yellow, if the pH drops the Methyl red changes the color to red.

What are antiseptics that we used

Mouthwashes

capsule stain

Negative stain - the structure of interest (capsule) remains colorless Uses acidic dye - Staining reagent Maneval's solution (acid fuchsin)

ground beef when cooked, Constructive or destructive?

Neither

Distinction between nitrogen reduction and denitrification

Nitrate reduction NO3- ->NO2- -> NH3 (ammonia) used by facultative anaerobes without the presence of oxygen. Denitrification NO3- -> bunch of steps N2-.

Isotonic

No net movement of water molecules, cell structure isn't affected by the tonicity of the environment.

Psychrophile

Optimal growth below 15C

destructive milks plated

Pasturized milk products

Exoenzymes

Peptidases exported from the cell and catalytically active outside the cell.

Describe the VP test

Pink ring signifies a positive result. This represents the use of the Butenol pathway. because the acetoin reachts with potassium hydroxide and alpha-naphthol. If it stays clear then it is a negative

gram stain (differential)

Primary stain - Crystal Violet Mordant - Gram's Iodine Destain - Acetone-Alcohol Secondary stain - Safranin

crystal violet stain

Primary stain, gets stuck in the cell wall (peptidoglycan

Destructive juice plated

Processed, frozen concentrate, fresh squeezed

Destructive processes

Produce food spoilage.

Desulfhydrase

Protein test: Another do KIA test

Gelatinase Test

Protein test: detects enzymatic breakdown of gelatin to polypeptides. and polypeptides to amino acids

cytochrome oxidase

Put the microbe on a slide and use oxidase reagent. oxidizes cytochrome C3 A dark purple color will show within 30 seconds.

Denitrification

Series of reactions to reduce nitrate to nitrogen gas (N2.) Other gaseous forms of nitrogen are also formed (NO and N2O.) Returns nitrate back to the atmosphere.

MRVP

Red ring=positive, acetone is produced and glucose is fermented using the butanediol pathway.

Tryptone

Red ring=positive, tryptophanase is produced and the organism can break down tryptophan into pryruvate, ammonia and indole.

KIA

Red=protein utilization Yellow=acid production and carbohydrate fermentation of glucose and/or lactose. Gas bubble= gas produced Black precipitate= desulfhydrase is produced

Halophiles

Require higher salt concentration, up to 20%.

Constructive processes

Result in desirable products and often involve fermentations.

2 bacteria involved in yogurt producion

S. thermophilus, lactobacillus bulgarious

Margin of a colony (6)

Smooth, Undulating, Lobed (almost villi coming out), irregular (rough), ciliate (lot's of white lines through it). filamentous (branchy)

darkfield microscopy

Species looks bright against a dark bacground.

flagellar motion

Straight lines or flagella tumbles.

Microbiology

Study of microscopic organisms that are below the limit of vision of the human eye.

What is P. polymyxa

The bacteria that produces the natural antibiotic.

Describe the Simmons Citrate test

The citrate starts out with a green medium. It changes to blue slant on top if the pH goes up and the citrate (acid) is broken down

Total viable Count

The direct quantification of the number of living cells in the culture- Enumerate with Colony forming Units/ (dilution factor*volume plated)

cellular arrangement

The patterns of cells

cell morphology

The physical appearance and behaviour of bacteria under a microscope including size, colour, gram staining and colonial appearance.

Nose piece

The rotating part of the microscope with the objectives.

Fine focus knob

The smaller knob that is used for precision focusing

zone of inhibition

The zone where bacteria can't grow around a given antibiotic. (small - resistant)

Antiseptics

Used for topical application to living tissues.

Immunofluorescent microscopy

Used to discover organisms that can fluoresce

phase contrast microscope

Uses refractory properties of light, creating a 3D image; it is good for visualizing cell organelles; "straw in water"

Normal microbiota

Usually inhabit our body and cause no harm under normal circumstances.

How does ELIZA work?

Wells are filled with wash buffer, then the first antibodies are put in, then they are wash with buffer, then the secondary conjugated antibodies are pipeted in. Then they are washed again. Then the substrate is put in. The positive results will show the conjugated secondary antibodies.

E test

Where a plastic strip with a concentration gradient are impregnated on one side.

Yeast VS bacterial Cell

Yeast: budding, large, and nucleus and other internal structures Bacteria: small, no budding, no internal structures

Glucose, lactose, and manitol

Yellow means acid production and carbohydrate fermentation. Bubble means gas production.

parafocality

ability to remain in focus when switching objectives.

in the precipitin test, what is present in the serum, "agent" of disease

anti-BSA

white precipitate

anti-BSA + BSA, how do you know that antibody-antigen interaction has taken place

primary antibodies

antibodies present in tested serum samples as result of an individual's exposure to a pathogen

acidic dyes

are negatively charged and work best at low pH

Arm of microscope

backbone of the microscope

Hats should be worn _______

backwards

gram positive

bacteria that have a cell wall less complex and contains more peptidoglycan than the cell wall of gram-negative bacteria. (Gram-positive bacteria are usually less toxic than gram-negative bacteria. and stains purple)

acid fast stain

binds strongly only to bacteria that have a waxy material in their cell walls ( wax prevents stains from gram stain) Primary stain: carbolfuchsin Mordant: turgitol & phenol Decolorizer: acid-alcohol Counterstain: methylene blue (Mycobacterium tuberculosis, Nocardia sp.)

What are the disinfectants and antiseptics we used?

bleach, lysol, iodine, 70% ethanol, H2O2m, isopropyl alcohol

Concentration equation

c.f.u./(volume plated)x(total dilution)

What is halotolerant?

can survive high salt concentration but doesn't require them.

hypotonic

cell could burst from an excess buildup of osmotic pressure, most bacteria, more turgid, lower concentration of solutes exists inside the cell, water will be drawn into the cell

Maneval's stain

cell is red and capsule is not stained

3 media classification

chemically, functionally, nutritionally

TSA

complex, rich, general purpose

PIA

complex, rich, selective Pseudomonas Isolation Agar

MacConkey

complex, rich, selective differential

BHI

complex, very rich, general purpose Brain Heart Infusion

Is cheeze creating constructive or destructive

constructive

simple stains

crystal violet, used to determine bacterial size, morphology, and cellular arrangement

classification of media MM1, MM1 without glucose, MM1 without sulfur

defined, minimal, selective

Heterotrophic

depend on an outside food source

Is ground beef constructive or destructive, when not cooked

destructive

Catalase Test

drop H2O2 and see if there are any bubbles, if there are bubbles there is catalase activity.

Are endospores or vegetative cells more likely to be heat resistant?

endospores.

Size of S. cerevisiae

eukaryote 5-10 micrometer

Elevation of colony (6)

flat, Umbonate (tiny hump on convex surface), Raised, hilly (rough), craterform (concave with the sides raised up), convex.

Order of ELISA

fluid sample, secondary antibody, substrate

secondary antibodies

generated to react specifically with the antibodies in the initial Ab-Ag complexes and allow detection of these complexes at concentration where precipitation or agglutination reaction normally do not occur

basic dyes

have a positive charge which is attracted to negative charge of bacterial cells

places where biofilms are

hospitals, anywhere

what helps endospore with high temp resistance

hydrophobic proteins in the endospore coat to maintain the low internal concentration of water

intermediate

indicates that the antibiotic may be useful under certain circumstances (effective in high concentration etc)

resistance

indicates that the organism causing the infection is resistant to the antibiotic and the antibiotic may not be the appropriate choice to treat this particular infection

susceptible

indicates that the organism causing the infection is susceptible to this antibiotic and the antibiotic may be the appropriate choice to treat this particular infection

Photoautotroph

inorganic compound carbon source, light energy source

Chemoautotroph

inorganic coupound carbon source, inorganic compound energy carbon source

all used glassware contaminated or not

items that must be disposed in disposal cart

hypertonic

loss of water from bacterial cell can result in plasmolysis, death of bacterium, higher concentration of solutes exists outside the cell, water will be drawn out of the cell

neutrophile

majority of bacteria have a pH optimum ranging from pH 6.5-7.5

How can a lower temperature affect generation time?

make it longer.

gram negative

more complex cell wall and contains less peptidoglycan ( Gram-negative bacteria are often more toxic than gram-positive bacteria. (appear pink- lose purple in gram stain) )

Acid stains tend to be ___________ because the color is carried by the negative ion.

negative stains

Is autoclaved milk constructive or destructive

neither

isotonic

no net movement of water molecules, cell structure not affected, equal concentration of solutes exists outside and inside the cell

Chemoheterotroph

organic compound as carbon source, organic compound as energy source

Photoheterotroph

organic compound carbon source, Light as energy source

Oculars

part of microscope you look into

Stage clip

part that holds the microscope slide in place

What antibiotic is produced by polymyxa, What is it more susceptible to

polymixins, gram negatives.

Size of E. Coli

prokaryote .5-2 micrometer

used microbe slides, toothpick, pipets, centrifuge tubes, swabs, broken glassware

red biohazard container

parfocality

remains close in focus as it switches from objective to objective, minor adjustments with the fine focus knob may be necessary

obligate aerobes

require oxygen

SEM

scanning electron microscope shows 3D picture

Goal of using KIA

simultaneous use of glucose an lactose in the medium, Also be used to detect proteins included in the medium

planktonic cells

single celled microbes

when ELISA is used to determine if an individual has been exposed to a particular disease, it detects the presence of

the antibody whose production was triggered by the pathogen

Virulence

the level of difficulty of a certain pathogen to penetrate the hosts defenses.

minimal inhibitory concentration (MIC)

the lowest concentration of an antibiotic that is needed to inhibit the growth of a particular organism under lab conditions, use Etest

Why do you invert plates

to prevent excess condensation

TEM

transmission electron microscope Shows inside of cell

Consistency of a colony (3)

transparent, tranlucent (blocks some light), opaque

wrappers, uncontaminated paper towels, gauze

trash bin

Cytochrome oxidase activity test

use a toothpick to transfer a large amount of cells from each of the pure culture plate to filter paper, add oxidase reagent purple indicates positive result

Catalase activity test

use an inoculation loop to add a visible amount of cells from each of the pure culture plates to the microscope slide add one drop of 5% H2O2 (bubbles if positive) (Bacteria produces catalase, which breaks down toxic H2O2 from aerobic respiration into H2) and O2.)

differential stain

used to more than one dye to stain an organism and show cell components.