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Microbiology Lab Practical Exam

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What is this piece of equipment called and how is it used?
Graduated Cylinder;
used for liquid measurements.

What is this piece of equipment called and what is it used for?
Beaker; cook with it

What is this piece of equipment called and what is it used for?
Flask, cook with it

What is this piece of equipment and what is it used for?
Weight Scales; for measuring weight

What is this piece of equipment called?
Bunsen burner

What is this piece of equipment called and what is it used for?
Inoculating needle; used for transferring bacteria when it must be stabbed.

What piece of equipment is this?
Depression Slide
(it is ground out in the middle)

What piece of equipment is this and how is it used?
Durham tube; glass tubing sealed on one end. Used in sugar fermentation test (traps gas bubble)

What are these called and how and when are they used?

Cover slips; used with depression slide to look at live bacteria.

What is this piece of equipment called and how is it used?
Spatula; a metal scoop used to measure out media

What is this equipment called and what would it be used for?
Test tube; used for growing bacteria in fluids or broths
What is media?
microbial food that may contain specific foods, pH buffers, minerals, vitamins, water and enrichments to meet the growth requirements of specific microorganisms
What are the Basic Media Types?
Broths-liquids
Gelatin-broth with gelatin added to become a semi-solid
Agar-solid, Jell-O-like
When mixing media to what place to we round up.
Always round up and measurements should go to the 10th of a gram.
(i.e. 1.4 grams instead of 1.38 grams)
The recipe says we need 8 g of powder to make 1 liter of Nutrient broth. How many grams of powder are needed to prepare 25 mL of Nutrient Broth?
8 g X 25 mL = 200 ÷ 1000 = 0.2g
Our recipe says we need 50 g of powder to make 1 liter of MacConkey II Agar. How many grams of powder are needed to prepare 16 mL of MacConkey II Agar?
50 g X 16 mL = 800 ÷ 1000 = 0.8 g
Our recipe says we need 20 g of powder to make 1 liter of Phenol Red Mannitol Broth. How many grams of powder are needed to prepare 35 mL of Phenol Red Mannitol Broth?
20 g X 35 mL = 700 ÷ 1000 =
0.7 g
Our recipe says we need 35 g of powder for 1 liter of Spirit Blue Agar. How many grams of powder are needed to prepare 41 mL Spirit Blue Agar?
35 g X 41 mL = 1435 ÷ 1000 =
1.435 g ---> 1.5 g
Our recipe says we need 55 g of powder for 1 liter of Kligar Iron Agar. How many grams of powder are needed to prepare 25 mL of Kligar Iron Agar?
55 g X 25 mL = 1375 ÷ 1000 =
1.375 g ---> 1.4 g

What piece of equipment is this?
Microscope

What part of the microscope is this?
Eyepiece

What part of the microscope is this?
Lamp

What part of the microscope is this?
Nosepiece

What part of the microscope is this?
Base

What part of the microscope is this?
Stage

What part of the microscope is this?
Substage Adjustment

What part of the microscope is this?
Objectives

What part of the microscope is this?
Fine Adjustment

What part of the micrscope is this?
Arm

What part of the microscope is this?
Diaphragm Lever

What part of the microscope is this?
Course Adjustment
What is the temperature setting for the refrigerator?
1 degree C
What is the temperature, psi, and duration for the autoclave?
121 degrees C and 15 psi for 15 minutes
What is the temperature range for the incubator>
25 degrees C to 37 degrees C
What is the temperature setting for the oven and what is it used for?
300 degrees C; used as a glass dish cleaner

What is the name of this equipment and how is it used?
Petri Dish; used to grow bacteria utilizing media (on the top lid)

What is the name of this piece of equipment and what is it used for?
Inoculating loop; used for transferring bacteria to the surface of media.

What equipment is being used to run this test?
Agarose Gel Electrophoresis

Based on the information which suspect cannot be excluded?

Suspect JT

What test utilizes this chemical to see shape and arrangement?
Negative or Background Staining
What media is used for the Negative (or background) stain.
What chemical is used for the negative or background staining?
India Ink
What are the possible shapes that can result from a negative or background stain?

cocci, bacilli, spirochute
What are the possible results for arrangement for the negative or background stain?

example: diplococcus

What is the name of the test these two chemicals are used for staining in?
Capsular Staining
What does a capsular stain test for?
The presence or lack of a capsule
What media is used to conduct the capsular stain?
None is used.
What chemicals are used to conduct the capsular stain?
Crystal Violet and India Ink.
What are the possible results of the capsular staining test?
A positive result will show a purple bacteria with a dark background. The area between these two will be a clear area. This is the capsule. A negative result will lack this clear region.
Are there any special genera associated with the capsular staining test?
What is a Smear Preparation?
It is the first step in the staining process in which bacteria is adhered to the slide so it won't wash off or become distorted during the staining process. This is done by placing water and the bacteria on the slide and allowing it to dry and then heat fixing it.
What test is conducted to determine the nature of the cell wall?
Gram Stain
What media is used in a Gram Stain?
What Chemicals are used in the Gram stain and in what order?
1. Crystal Violet
2. Gram's Iodine
3. Ethly Alcohol
4. Safranin Red
What are the results of the Gram Stain test?
A positive result is purple
A negative result is pink
Are there any special genera that may be tested for with the Gram stain?
What test will determine if a bacteria produces an endospore?
The Spore Staining (Schaeffer-Fulton Method)
What media is used for the Spore staining test?
What chemicals are used for the spore staining test?
Malachite green
Safranin red
What is being tested for when conducting the spore staining?
The presence or lack of spores or endospores
What are the results of the spore staining test?
If spores are present, the malichite green will stain the interior of the spore green. The outer portion of the spore will stain pink from the safranin. If the result is negative no green color will be observed.

What test utilizes a depression slide and slip covers attached by vaseline and viewed under oil emersion?
Motility Determination Test
(Hanging Drop Method)
What is being tested for when conducting the motility determination test?
Whether the bacteria is capable of moving (motility)
What chemicals are used in the motility determination test?
What media is used in the motility determination test?
What tests are used to determine morphological characteristics?
Negative Staining (Background staining), Capsular Staining, Gram Staining, Spore Staining, and Motility Determination
Which genera(s) includes spore forming as a major characteristic?
Bacillus and Clostridium
________ ____________
What tests are used to determine cultural characteristics?
Nutrient Agar Culture, Nutrient Broth Culture, Gelatin Stab Culture, Nutrient Agar Slant, Blood Agar, and Oxygen Requirements,
What test is utilized to determine the colony characteristics of a bacteria>
The Nutrient Agar Culture
What Media is used to test for the colony characteristics of a bacteria?
Nutrient Agar Plates
For which bacteria type is the method of Negative Staining also used and why?
Spirochetes because they are difficult to stain.
What media is used for a smear preparation?
Bacterial Broth or Bacterial Agar.
What chemicals are used for the Nutrient Agar Culture?
What does the Nutrient Broth Culture test for?
Where the growth occurs and how its particles present when suspended.
What media is used for the Nutrient Broth Culture?
Nutrient Broth
What chemical(s) are used for the Nutrient Broth Culture?
What are the possible results for the Nutrient Broth Culture?
Growth can be surface, subsurface or sediment.
Suspended can be turbid if cloudy, granular if small particles are seen, or flocculent if masses are floating around.

Name the test that utilizes this.
Gram Stain

Name the test that utilizes these.
Spore Staining

What is this test?
Nutrient Stab Culture
What are the possible results of the Nutrient Agar Culture?

Shape: Round, Filamentous, or Irregular

Elevation: Flat, Raised, Convex, Drop, Hilly, or Ingrowing

Margin Edge: Smooth, Wavy, Lobate, Irregular, or Ciliate
A gelatin stab culture determines what?

#1 liquefication: If the organism produces gelatinase(gelatin digestive enzyme)
#2 if no liquefacation occure a growth partern is given. Filiform, beaded, arboresent
What is being tested in a Gram stain?
positive or negative stain thickness of peptidoglcan
What are the results of Gram staining
Postive= Violet
negative= red/pinkish

No cell wall= red
A nutrient Agar Slant determines what characteristics

#1 color in reference to pigments
#2 form of how organisim will grow. example Filiform, Echinulate, Effuse, arborescent.
What does the media blood Agar determine?

If the bacteria is hemolytic or non-hemolytic. hemolytic bacteria distroys red blood cells in media making either clear zones(beta) or Green zones (alpha)
What special genera are associated with Blood Agar
Streptococcus and staphylococcus
To determine Oxygen requirments what medium is used?
Thioglycollate
What results are determined by an oxgen requirement test?

Aerobic(growth on surface) Microaerophilic (surface and subsurface) facultavie spotty growth all over medium, Anaerboic (groth at bottom only)
What characteristics are determined by starch hydrolysis and what will the results show

If the bacteria is capable of hyrolyzing starch, Iodine will turn agar purple. clear zones indicate + starch hydrolysis
What special genera is used for starch hydrolysis
Baciullus subtilis a positive control for the test should show clear zones
What chemeicals are used for starch hyrdolysis
Grams Iodine
What media is used for starch hydrolysis
Starch agar
What does the Casein hydrolysis test determine
If the bacteria is capable of hydrolyzing casein, a milk protien
What media is used for the Casien hydrolysis test?
Skim milk agar
What chemicals are used in the casien hydrolysis test
none.
What special genera is used in the Casien hydrolysis test?
Bacillus subtilis a postive control will show clear zones
What is being tested for in a fat hydroylis test?
if the bacteria is capable of hydrolyzing fat
What media is used in the fat hydroylis test?
spirt blue agar
What special genera is used for fat hydrolysis?
Bacillus subtilis for a postive control will show blue precipitate around the growth
what does the Methyl Red Test determine?
If the bacteria will produce large amounts of acid(lowers PH)
What media is used in the Methyl Red Test?
methyl red-vp broth
What special genera are used for methyl red test
Escherichia, salmonella, Proteus, Aeromonas
How the results of the methly red test read?

Postive will show red to orangle color. negative will appear yellowish
What does the Urease test determine?
if the bacteria used can break down urea
how are the results given for a Urease test?

Postive will appear pinkish to purple. Negative will appear light orange.
What is the special genera used in the Urease test?
Proteus vulgaris a postive control will appear pinkish to purple in color
What does the Indole test determine?
If the bacteria will hydrolyze tryptophan, producing indole and pyruvic acid
what media is used in the indole test
Tryptone broth
what chemicals are used for the Indole test?

Kovacs reagent
how will the results read for the Indole test?

Positive will show a red ring at the surface. Negitive a clear or yellowish ring.
What does the Catalase test determine?
If the bacteria can produce catalase.
what media is used during the Catalse production test?
Agar slant or plates
What chemicals are used in the catalase production test?
Hydrogen peroxide
How are the results given for the Catalase production test
when hydrogen peroxide is added the culture will bubble indicating Postive catalase production
What does the Durham tube Sugar Fermentation test determine?
Which carbohydrates may be utilized for energy. (which sugars are fermented).
What media is used in the Sugar Fermentation test?
Phenol red broth,
glucose
lactose
mannitol
saccharose
how are the results of the sugar fermentation test shown

The media will turn from red to yellow in the presence of acid
The results should record the presence or absence of both the acid and gas. Gas will show bubble in durham tube.
What does the hydrogen Sulfide production test determine?
If hydrogen suilfide is produced.
What media is used in the hydrogen sulfide test?
Kligler's Iron agar stabs(contains ferrious sulfate, will react with hydrogen sulfide.
how do the results of the hydrogen sulfide test read?

Formation of a dark black color a pereciptitate of iron sulfide
What special genera is used in the hydrogen sulfide test?
Proteus vulgaris for postive control.
What does the Citrate Utilization test determine?
If the bacteria can utilize citrate a a sole source of carbon
What media is used for the citrate test?
Simmons citrate agar slant
how do the results read for the citrate test?

It will change from green to a deep blue if growth occurs. No growth indicates a negative test.
What special genera is used in the citrate test?
Bacillus subtilus is a control positive.
What are the possible results of the Fat Hydrolysis Test?

A blue precipitate around the growth is an indication that fat is hydrolyzed.
What media is used for the Gelatin Stab Culture?
Nutrient Gelatin
What chemical(s) are used for the Gelatin Stab Culture?
What special Genera are associated with the Gelatin Stab Culture test, if any?
What media is used for the Nutrient Agar Slant?
Nutrient Agar Slants
What Chemical(s) are used for the Nutrient Agar Slant?
What media is used in the Urease test?
Urea Broth
What magnification power is the scanning objective lens on a microscope?
4X magnification + eye X 10= 40
What magnification power is the Low power lens on a microscope?
10X magnification + eye X 10
What magnification power is the High power lens on a microscope?
40X magnification + eye X 10=400
What magnification power is the Oil emmersion lens on a microscope?
100X magnification+ eye X 10= 1000
How is a positive motility determination test?
Motility is confirmed by the quick darting action of the bacteria through the liquid.

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