Micro lab quiz 1

this is created by Hannahlerner144 in quizlet. I just imported to be able to study them with the learn

Autochthonous

A microorganism that is native to a location.

Allochthonous

referring to a microorganism that is not native to a site

Aerobe

A microorganism that needs oxygen to live and grow

Anaerobe

a microorganism that can thrive in the absence of oxygen

Facultative anaerobe

microorganism that can live with or without oxygen

Methods for growing anaerobes (4). What are each of these?

anaerobic chamber (filled with gas with no O2) or glove box (N, Co2, H), anaerobic jar (inexpensive), roll tube (small test tube with anaerobic mix) and thioglycolate broth tube (less sensitive to o2)

Microbiome

kinds of microorganisms normally found at some environmental site

What agar do we use?

BHI (brain heart infusion) that is made from BH in cows

Why do you not want to swab your saliva when you swab your throat

different bioime than the back of your throat

What gave the most colonies and why? (testing env)

Throat and sink bc most water and nutrients are present. Microbes are common.

What is recovery influenced by?

Conditions: Water, food, temp, pH, oxygen

What colony type and color were from each sample

Throat- beige, yellow, small
Armpit- White, small
Sink- Grey/yellow, medium
Air- Black, large

Where are these three samples found: Bacillus subtilis, staphylococcus epidermidis, and escherichia coli?

BS: Nature and soil
SE: Human skin, can be assoc w disease
EC: in GI tract, asssoc w disease

What is the objective and power for oil immersion?

100x objective, 1000x power

Motility, clusters and shape for
BS
SE
EC

BS- Large rod, + motility, singles pairs threes and fours
SE- Small spheres, - motility, singles, pairs and clusters
Ec- small rod, + motility, singles pairs

Why is it hard to see bac without staining?

Transparent

what does it mean to be motile

Can swim because have a flagella
SE do not have a flagella

What is Brownian motion?

the erratic random movement of microscopic particles in a fluid, as a result of continuous bombardment from molecules of the surrounding medium. NOT motility but still important tto see

What is a chain

Long series of cells together, more than 4

Colony

a large group of cells produced from one cell (identical)

Pure culture

Culture of only one strain

Working under a flame

microbiologists frequently work under the flame of a gas burner which provides an updraft so microbes in the air don't fall inside materials such as tubes and plates

What are serratia marcescens

look like ecoli but they are in nature, they like sinks and showers

How do you inoculate broth?

flame sterilize--remove capof culture-obtain culture and add cap back on-move cap sterile tube-plunge loop w bac in sterile broth

How do you inoculate agar slant tube?

Agar inside tube touched and moved back and forth up from the base of the slant

How do you inoculate a streak plate?

4 quadrants- draw on paper and show where singles should be.

What is the main goal of streaking the plate?

Obtain isolated colonies/ pure culture

Explain results of each medium after inoculation and incubation

Broth- cloudy
Slant- film grew (in ecoli is wt/beige)
Plate- Red colonies grew (serratia)

Aseptic technique

precautions taken to prevent contamination of the environment.

Mixed culture

A container growing two or more different, known species of microbes.

Contaminant

undesirable mutants that can cause unwanted results
Different colonies

What was seen on the plate that was undesired?

White spots were seen that were mutants.

Stain or Dye

An organic compound with a chromophore group (gives color) and a auxochrome group (gives a positive or negative charge)

Why is it called a simple stain

Easy to use, only uses 1 dye

What are basic dyes

crystal violet, safranin (red), malachite green, and methylene blue.

Why are basic dyes used

Basic dyes are used in staining because cells have chemical groups with a negative charge.

How do you heat fix your sample? Why do you do this?

passing the dried sample face up through the flame of a gas burner for 2-3 sec. Do this so you can stick the bacteria to the glass.

How long do you let the dye sit

1 min

What shape are BS, SE and EC

BS- large rod
SE- spherical clusters
EC- small rod

How do you prepare a smear of bacteria that was grown on an agar surface?

A drop of water is placed on a slide and a small amt of bacteria is gathered with inoculating loop then mixed tg on the slide.

What do you have to avoid when preparing a smear of bacteria from agar?

Avoid making a paste.

Autoclaving

steam under pressure in a chamber
121C, 15 lbs per square inch, 15 minutes minimum
Will destroy ALL microbes
doesn't make a mess or need chemicals

Pasteurization

heating in a water bath at 63C for 30 minutes.
Destroys most pathogens
does not destroy non pathogen

Dry heat

Baking 160 to 180 C for 1-2 hours will destroy all microbes
"oven"

Filtration

sterile nitrocellulose filters with .45 um pores are good to remove cells from heat sensitive liquids, such as antibiotics
Holes are small

UV light

UV lamps are frequently used to disinfect lab surfaces

Ultrasound

High frequency sound used in machines to clean instruments or break cells but DOESNT STERILIZE

What 3 tubes needed for common phys agents experiment?

Control
Sonicated
Autoclaved

Of the 3 tubes which will have growth

Control and Sonicated

Disinfectants

Phenols, chlorine, and glutaraldehyde used to kill living cells

Antiseptics

Ethanol and Isopropanol

Difference between antiseptics and disinfectants

Antiseptics can be used on the body without causing skin damage and are more gentle.

Chemicals that remove microorganisms

Soaps and detergents, remove dont kill

Chemical used for sterilization

ethylene oxide gas

Show how to make a lawn

How do we test for antimicrobial activity

create a lawn then spot with detergent, NaCl, Alcohol and bleach

What inhibition zone did each chemical agent cause?

Detergent-0
NaCl- 0
Alc- +2
Bleach- +4

What does each number mean regarding inhibition zone

0- nochange
+4- total killing

Differential Staining Techniques

gram stain and acid-fast (or Ziehl-Neelsen stain)

Structural staining techniques

spore stain and flagella stain

Cell biochemicals that affect staining

peptidoglycan, lipids and capsule polysaccharides

Why do we want an old culture of bacillus subtilis?

spores

Negative stain reagent

india ink

Gram stain reagents

crystal violet, safranin, iodine and ethanol

Spore stain reagents

malachite green and safranin

What is the order of staining in special staining

1. Prepare smear w ecoli and s epidermidis
2. Stain w crystal violet for 1 min(primary stain)
3. Iodine stain (fixative) for 1 min
4. Decolorize with EtoH for few seconds
5. Counterstain w safranin

Gram positive vs gram negative

Gram positive is purple
negative is red

is ecoli/ s. epidermidis gram positive or negative

Ecoli is gram negative and red, while s.epidermidis is gram positive and purple

Why do gram positive stay purple

crystal violet- iodine complex cannot wash out with ethanol. S. Epidermidis must have low lipids and high carbohydrates to stay.

What levels of lipid/carbs will be gram pos/neg

High lipid, low carb- gram negative (red)
Low lipid, high carb- gram positive (purple)

How do you get stain in the spores

steam the slide with malachite green for 4 mins periodically
then counterstain with safranin

What type of stain is a spore stain and why?

structural stain, since endospores are components of cells, spores are resistant so they must be stained

Negative stain

Stains the background rather than microbes, works well for cells that have large capsules and do not stain well

K. pneumoniae have what?

have a mucoid capsule

What is the purpose of acid-fast staining?

Acid fast shows cells with wax such as mycobacteria, this is how they discovered TB.

colony count

Samples of diluted culture are spread over surfaces of agar in Pertri plates. After incubation, the number of colonies and the dilution factor can be used to calculate cell concentration. Only numbers between 30 and 300 are considered valid numbers for counting.

Petroff-Hausser counting chamber

an engraved grid on a microscope slide is used for counting cells in a known volume.

Coulter counter

cell number and size can be determined in this machine which measures electrical resistance

Spectrophotometer

the cell concentration can be determined in this device by measuring the light absorbed

Cell Mass

dry or wet weight gives an estimate of the number of cells

Metabolic Methods

oxygen consumed or carbon dioxide produced can indicate microbial growth

what 5 tube dilutions will be preformed?

0, 10^-2, 10^-4, 10^-6, 10^-8

How much of the previous cells will be transferred from one tube to the next?

.1 mL

Equation to determine cell concentration

(\# colonies x 10 x 1) / Tube dilution \= cell concentration(cfu/mI)

Calculate the CFU/mI of 35 colonies/plate on the 10-6 dilution plate

35 x 10 x 1/10^-6 \= 3.5 e 8 CFU/MI

Does this assay method detect every cell body spread on a plate and why?

No, some cells are dead. Pairs chains and clusters make one colony.

Methods of measuring cell concentration that may be faster than colony count assay.

Spectrophotometry

What major illnesses are caused by staphylococci?

boils, scalded skin syndrome, toxic shock syndrome, food poisoning (staphylococcus aureus)

major diseases caused by streptococci?

contagious skin disease, sore throat, rheumatic fever, glomerulonephritis (streptococcus pyogenes: flesh eating bacteria), pneumonia (streptococcus pneumoniae)

Beta-hemolysis in staphylococci?

this activity produces the lysis of red blood cells in agar plate medium. Blood agar is prepared by mixing 5% sterile defibrinated sheep blood with a nutritional blood agar base that is cooled to 45C after autoclaving. Medium is dispensed into tubes or plates.

Hemolysis in streptococci

lysis of red blood cells in agar (beta-hemolysis), conversion of red blood cells to green (alpha-hemolysis), no effect on red blood cells (gamma-hemolysis)

Selective medium

medium to prevent growth of some species but permitting others to thrive

Differential Medium

medium that enables the investigator to distinguish one species from another. this is usually done through use of a chemical reaction which causes the color of the medium or colonies to change

*Major Types of Test Media for Gram Positive Staphylococcus

mannitol, staph 110 agar, citrated rabbit plasma

Mannitol Salt Agar

This plate medium helps to identify S. aureus because the species often ferments this carbohydrate (yellow). Phenol red is used as a ph indicator. Sodium chloride helps to inhibit other microbes. S. epi is pink

Staphylococcus 110 agar

This plate medium contains gelatin because some strains of S. aureus can produce gelatinase. Sodium chloride helps to inhibit other microbes. Lactose and mannitol are added as nutritional sugars.

Citrated Rabbit Plasma

a few drops of plasma with added citrate in a small tube or on a slide can test for the enzyme coagulase because it will cause the liquid to clump or gel. Only S. aureus makes the enzyme but not S. epidermidis

Mediums for Gram-positive streptococcus

Bile Esculin Azide agar, taxos a disc, and taxos p disc

Bile Esculin Azide Agar

Plates of this agar are used for the selection and enumeration of fecal streptococci. The ingredients include: oxgall, esculin, ferric ammonium citrate and azide as a selective agent.

Taxos A Disc

this disc which contains bacitracin is used to distinguish group A beta-hemolytic streptococci (S. pyogenes) which is bacitracin sensitive from other beta-hemolytic streptococci on an agar plate