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Flashcards in Midterm Deck (94):

What methods are used to see motility and how motility is determined?

- Hanging drop technique or SIM tubes


How to determine if motile in SIM tubes

- if the bacteria turns yellow gelatin black and strays away from the stab line/spreads throughout the tube



- for carrying the microscope
- supports the lenses


What do you observe when looking at the growth of bacteria in a nutrient agar plates?

- Colony size, color, form, elevation, and margin


light intensity dial

- adjusts current to lamp


Filter Paper Disc Method Results

- a zone of inhibition (no growth around disc) is indicative microbicidal activity against the organism
- absence of a zone indicates that the chemical was not effective against the test organism
- measure from the edge of disc to the edge of growth (diameter mm)
- identify as R, I or S (resistant-lots of growth, intermediate-some growth, or sensitive-no growth)


Inoculating procedure

1. sterilize the loop with the flame
2. remove cap of bacteria tube and flame the tube 3x before inserting the loop
3. get a loopful of culture
4. heat the mouth of the tube and replace the cap


Blood Agar

- enriched and differential
- gram positive and gram negative
- gamma-hemolytic(no change red), alpha hemolytic(greenish halo around growth) and beta hemolytic (clear zone surrounding the colonies)


Concentration of Salt affect on bacterial growth (.5%, 5%, 10% and 15%) in E.coli, S.Aureus and H. salinarium

- no bacteria was able to tolerate 15% and did not grow
- as the salt concentration increased, the amount of growth did as well

1. E coli- highest can tolerate is 5%
2. S Aureus- highest can tolerate is 10%
3. H salinarium- highest can tolerate is 10%


Starch hydrolysis Test

- ability to degrade polysaccharide starch, lipid tributryin and proteins casein and gelatin
- Starch Plate
- add several drops of grams iodine if there is a clear zone positive test result


How to make a smear using a liquid media

1. place 2 loopfuls of liquid culture on the slide with a sterile loop
2. spread the bacteria in a circle/target area
3. allow the smear to air dry
4. heat fix


How to make a smear using solid media

1. get a loop of water and smear on targeted area of slide
2. remove the bacterial culture and mix with water
3. allow to air dry
4. heat fix to kill bacteria


Nitrate Reduction Test

- ability to reduce nitrates to nitrites
- nitrate reduction broth with Durham tube
- if no gas add 5 drops of Baritts A then 5 of B, no color means negative
- to confirm negative by adding a pinch of zinc powder
- would have turned deep red if positive


Zone of Inhibition

- an effective agent will inhibit bacterial growth and measurements can be made of the size around the disc
- measure from edge of the disc to the bacterial growth (mm)
- indicate whether resistant, intermediate, or susceptible


What is the purpose of a gelatin stab and why is it important?

- the purpose is to determine the organisms ability to produce proteolytic enzymes
- a positive result will liquify the gelatin


What type of microscope do we use?

- brightfield compound microscope


Decolorizing agent affects gram positive and negative cells differently because

1. the concentration of lipids
2. the thickness of the peptidoglycan layer in bacterial cell walls


Facultative anaerobes

- can grow in the presence or absence of oxygen
- particles throughout tube, but condensed at top


Micrococcus Luteus was ______

- non motile


Phases of bacterial growth in a batch culture

1. Lag Phase
2. Exponential Phase (Log)
3. Stationary Phase
4. Death Phase


Why is heat applied in an endospore stain?

- to drive the stain into the endospore because most endospores are impermeable to staining



- growth from 7-14
- optimum growth at 10.5 basic


Aseptic technique

- used to exclude contaminants


Citrate Utilization Test

- ability to ferment citrate as their sole source of carbon
- Citrate agar slant
- if positive green color changes to blue


Effectiveness of Hand Scrubbing with alcohol vs soap technique used

1. Divide TSA plate into 4 sections
2. rub thumbs together
3. press pad of unwashed thumb in A, then in B
IF testing ALCOHOL
4. unwashed right thumb in C, 5. put thumb in tube of alcohol and press in D
IF testing SOAP
4. blot pad of right thumb on paper towel, place in C
5. wash right thumb with soap then press in D



- a substance produced by a microorganism that inhibits growth of other microorganims


Process of ammonification in the nitrogen cycle

- liberates ammonia by deamination of amino acids or catabolism of urea to ammonia
- after nitrification can occur to convert ammonia to nitrate



- populations or communities of microorganisms that attach and grow on a solid surface that has been exposed to water
- composed of pillars and channels through which water can flow, bringing nutrients and taking away wastes
- more resistant to antibiotics and more difficult for immune system to destroy


Why is it important to heat fix ?

- to kill the bacteria
- preserves microbes with minimal shrinkage or distortion when stained


How to interpret effectiveness of Hand scrubbing with alcohol vs soap

Percent reduction=( (colony count in 1st pressA or C) - (colony count in 2nd press B or D) ) / (colony count in 1st press A or C)


What is the total magnification equation?

- Total Magnification = (Magnification of objective lens) x (magnification of eye piece lens)


Gram positive cells color and how does cell wall structure determine how it stains

- purple
- has a thick peptidoglycan layer that has a retention factor making the primary stain color hard to remove


Streak plating technique Purpose

- to isolate colonies


Filter Paper Disc Method Procedure

- requires the heavy inoculation of an agar plate with the test organism
- sterile color coded filter-paper discs are impregnated with different antiseptics and spaced on the agar plate
- using pour plate method melt the agar and add the liquid bacteria to it
- dip the disc in your antiseptic (betadine) place firmly on the agar


What do you observe when looking at the growth of bacteria in a nutrient broth?

- Growth on surface, subsurface, and bottom of tube


What should the microscope lens be cleaned with after oil immersion?

- lens paper


Phenylethyl Alcohol Agar (PEA)

- selective medium
- gram positive


Methyl Red Test

- presence of stable acids ferment glucose
- MRVP broth
- add 5 drops of methyl red indicator, if positive tuns red, if negative turns yellow


Carbohydrate Fermentation Test

- to see if a bacteria is a fermenter of carbohydrates
- carbohydrate broth is used with Durham gas tube
- positive test results are if the initial phenol red turns yellow and look for gas presence in tube


Endospores are formed by which genera

- bacillus and clostridium


Filter paper disc method Purpose

- to test the effectiveness of an antiseptic/disinfectant


Ultraviolet Radiation as a microbial control agent

- nonionizing radiation between 15 and 400nm
- most lethal (UVC range) are biocidal at 200-290nm



- growth from 3.5-10.5
- optimum growth at 7 neutral pH


Antimicrobial drugs

- antimicrobial chemicals used internally to inhibit microorganisms, whether natural (antibiotics) or synthetic (man made)


Oxidase test

- cytochrome oxidase activity
- TSA plate (inoculate each specimen on half of the plate)
- add drop of oxidase reagent to each side, if turns purple positive


Indole Production Test

- to see if they can degrade the amino acid tryptophan
- SIM agar is used
- add 10-12 drops of Kovacs reagant, positive if a red ring on surface forms


Ocular Lens

- eyepiece
- remagnifies the image formed by the objective lens


What do you observe when looking at the growth of bacteria in a nutrient gelatin deep (stab)?

- Check for liquefaction or no liquefaction and the type
- patterns


Streak plating technique steps

1. streak the first sector, then flame loop
2. turn the plate and streak through edge of first sector and streak second sector
3. flame loop, do the same for the third sector
4. streak remaining area of agar surface


Kirby-Bauer Method Purpose

- evaluates the sensitivity if bacteria to certain antimicrobial drugs



- growth at pH of 0-7
- optimum at 3.5 ish acidic pH


Hydrogen Sulfide (H2S) Test

- ability to produce hydrogen sulfide from sulfur containing amino acids or inorganic sulfur compounds
- SIM tubes
- if you see black ferrous sulfide present H2S is present
- motility if moved away from the stab line



- focuses light through the specimen


What is Resolving Power

- Ability to completely separate two objects in a microscopic field


Gram negative cells color how does cell wall structure determine how it stains

- pink
- has a thinner and less cross linked peptidoglycan layer that releases the unbound CV-I complex and leaves it unstained


How is the hanging drop test performed?

1. Spread vaseline around a depression slide
2. place bacteria culture on coverslip
3. lower the depression slide onto the cover slip, seal
4. flip slide over so the culture adheres to coverslip
5. Use 400x magnification to view


Obligate anaerobes

- require absence of free oxygen for growth
- particles condensed at bottom


How did we examine the germicidal effects of UV light on S aureus and B subtilus?

- we inoculated a plate half with each bacteria
- then we covered half of the plate with a paper towel
- we exposed the plate to UV light at different time lengths
- the covered part grew substantially more than the uncovered area


which organism should have been able to grow at the highest salt concentration?

- H. salinarium is extremely halophilic


Acidic Stain

- if it is a negative ion/chromophore (anion)


Proteus Vulgaris was _______

- motile



- to hold the slide


Urease Test

- ability to degrade urea using urease
- Urea agar
- positive result will turn a bright pink color



- supports the microscope


Steps in Gram Staining

1. Primary Stain- crystal violet (all cells are purple)
2. Mordant- Grams iodine (intensify color of the stain)
3. Decolorizing Agent- Ethyl Alcohol (makes gram negative colorless)
4. Counterstain- safranin (stains gram negative cells pink)


Basic Stain

- if it is a positive ion/chromophore (cation)
- most bacteria are stained with this



- controls the amount of light entering the condenser


Negative Stain

- stains the background instead of the bacteria
- the bacteria will appear clear against a stained background
- use Maneval's A


What do you observe when looking at the growth of bacteria in a nutrient agar slant?

- Amount, color, opacity, form, consistency


Voges-Proskauer Test

- presence of acetoin to ferment glucose
- MRVP broth
- add 10 drops of Barritts A reagent and 10 drops of Barritts B reagent, shake every 3 min for 15 minutes
- Positive result is formation of a deep rose color


Acid Fast Stain Procedure

1. Primary Stain- carbolfuschin (red)
2. Decolorizer- acid alcohol (nonacid fast colorless)
3. cover with Counter Stain-
methylene blue (non-acid fast blue)



- light source


Bacterial capsules

- polysaccharides that are water soluble and uncharged
- play a vital role in virulence (disease causing ability)


Fine focusing knob

- used for focusing with high power and oil immersion
- changes the distance between the objective lens and the specimen


procedure to detect ammonification

- test soil for the presence indicates process of ammonification via soil microbes
- use peptone broth, soil
- after incubation test using Nessler's reagant and spot plate
- aerobic mud will be brownish and anaerobic mud will be black


Kirby-Bauer Method Process

- disk diffusion method
- Mueller Hilton agar is used
- place four discs with different concentration of antimicrobial drugs on plate
- measure the zone of inhibition and rate using (R,I or S)



- resting bodies that do not metabolize and are resistant to heating, chemicals and many harsh environmental conditions


Aerotolerant Anaerobes

- do not use oxygen, but are not killed by the presence
- growth even throughout


What genera of bacteria are acid fast and why?

- Mycobacterium
- because their cell walls contain a waxlike lipid called mycolic acid which renders the cell wall impermeable to most stains


Bacterial cell morphologies

1. Bacillus, or rod
2. Cocci, or round
3. Spirochete, or Spiral


Endospore staining Results

- stains any present endospores green
- stains bacterial cells red


Bacterial Growth curve

- used in a batch culture
- log of the number of cells per mL vs time
- measure the absorbance vs incubation time minutes
- in different dilutions


How does UV radiation affect DNA

- induces thymine dimers in DNA, resulting in mutation
- mutations result in death of cells
- exposure to UV reduces the amount of bacterial growth significantly especially over elongated periods of time


Casein hydrolysis

- ability to undergo proteolysis in the presence of proteases
- Skim milk plate
- if positive there is a clear zone


Coarse focusing knob

- used for focusing with low power objectives (4x and 10x)
- moves the lense or stages longer distances


Body tube

- transmits the image from the objective lens to the ocular lens


In tubes where is the high vs low oxygen concentrations located

- high is at the top of the tube
- low is at the bottoms of the tube


Simple Staining

1. Cover a smear with methylene blue and leave it for 30-60 seconds
2. wash it off with warer
3. blot it to dry
4. observe cell morphology, size and arrangement


Obligate aerobes

- require oxygen for growth
- particles at the top



- require limited amounts of Oxygen for growth
- particles mostly at the top and a few below


Oil immersion lense

- highest magnification and must be used with immersion oil (100x magn)
- It is used because it maintains continuity between the sample and the objective lens
- limits loss of light due to refraction.


Catalase Test

- ability to degrade hydrogen peroxide using enzyme catalase
- nutrient agar slant
- add hydrogen peroxide to growth on slant, if bubbles positive


Schaeffer-Fulton Endospore Staining Procedure

1. place absorbent paper over the smear
2. Primary stain- malachite green
3. steam the slide for 5 minutes
4. wash with water
5. Counterstain- cover the smear with safranin for 30 seconds
6. wash the smear with water and blot it dry


MacConkey Agar (MAC)
- functional type
- type of colony growth

- selective and differential
- gram negative grows
- lactose fermenters(red colonies) vs nonlactose fermenters (transparent colonies)