Lab Exam Flashcards
Motility Test
Motility Agar deeps are used to test motility. There is a pink dye within the media to show growth in the agar. A sterile needle is used to inoculate this media rather than a loop.
Positive Motility Test
fanning out away from the stab line. This can also make the tube look cloudy.
(Flagella present.)
Negative Motility Test
No fanning out from the stab line. (No flagella likely.)
Catalase Test
Aerobic respiration results in the formation of hydrogen ions (H+) which are converted to toxic hydrogen peroxides (H2O2) as part of the electron transport chain. The Catalase enzyme breaks down the peroxides into non-toxic H2O and O2. This enzyme is needed in order for a cell to exist in the presence of oxygen. Therefore, most bacteria are catalase positive with the exception of bacteria that cannot carryout aerobic respiration (i.e. obligate anaerobes).
Catalase Test Reagent
Hydrogen peroxide
Catalase Test Positive
Bubble formation after placing a drop of hydrogen peroxide on the
colony on a glass slide. (Catalase enzyme present.)
Catalase Test Negative
No bubble formation after placing a drop of hydrogen peroxide on
the colony on a glass slide. (No catalase enzyme detected.)
Nitrate Reduction Test
The ability to reduce nitrate to nitrite is commonly used to identify bacterial unknowns. The
media contains peptone and beef extract to support growth, as well as potassium nitrate. If the
organism can reduce the nitrate, it will react with the reagent.
Nitrate Reduction Test Reagents
PABA, a white powder.
Positive Result for Nitrate (NO3-) → Nitrite (NO2-)
Yellow, pink or red color change after the addition of the Paba.
Negative Result for Nitrate (NO3-) → Nitrite (NO2-)
No color change after addition of the Paba.
Glucose Test
Specifically tests for GLUCOSE breakdown, just because an organism breaks down one sugar doesn’t mean it breaks them all down. The potential outcomes are (+/+), (+/-), or (-/-). The first + or - is sugar breakdown (color change). The second + or - is gas production.
Durham tube
A small tube inside the larger tube of a glucose test. It is upside down and is there to catch any gas that is produced by the organism’s breakdown pathway.
Glucose Test: (+,+)
Yellow tube with gas bubble. (Glucose breakdown, gas produced)
Glucose Test: (+,-)
Yellow tube without gas bubble. (Glucose breakdown, no gas produced)
Glucose Test: (-,-)
Red tube, without gas bubble. (No glucose breakdown, no gas produced)
Differential Media
Colonies may be different colors based off of some
characteristics.
Selective Media
Media may inhibit growth of certain bacterial species.
Mannitol Salt Agar (MSA)
It selects for salt tolerant (or Gram positive) organisms, and differentiates based off the fermentation of mannitol. Growth (selection) is the first + or - and the color change (differential) is the second + or -.
Mannitol Salt Agar Outcome: (+,+)
Growth of organism, yellow agar color. (Salt tolerant organism, can ferment mannitol.)
Mannitol Salt Agar Outcome: (+,-)
Growth of organism, pink agar color. (Salt tolerant organism, cannot ferment mannitol.)
Mannitol Salt Agar Outcome: (-,-)
No growth of organism, pink agar color. (Not salt tolerant.)
MacConkey Agar
Identities lactose fermenting, Gram-negative enteric organisms, and for inhibiting growth of Gram-positive organisms (selective). Bacterial colonies that can ferment lactose turn the medium any variation of red (differential). This red color is due to the pH
indicators response to the acidic environment created by fermenting lactose. Organisms that do not ferment lactose do not cause a color change.
MacConkey Agar Outcome: (+,+)
Growth of organism, pink color change. (Gram negative organism, can ferment lactose.)
MacConkey Agar Outcome: (+,-)
Growth of organism, no pink color change. (Gram negative organism, cannot ferment lactose.)
MacConkey Agar Outcome: (-,-)
No growth of organism, no color change. (Gram positive organism.)
Bile Esculin
Some bacteria have the ability to survive in the presence of bile and hydrolyze esculin. The bile salts inhibit the ability of
certain bacteria to grow. Only bacteria that can tolerate bile salts grow on BEA. Differentiation of species able to grow on this media relies on the hydrolysis of esculin to form a dark brown or
black color change.
Bile Esculin Outcome:(+,+)
Growth of organism, black color change. (Bile salt tolerant organism, can hydrolyze esculin.)
Bile Esculin Outcome:(+,-)
Growth of organism, no color change. (Bile salt tolerant organism, cannot hydrolyze esculin.)
Bile Esculin Outcome:(-,-)
No growth. (Organism cannot grow in the presence of bile salts.)
Triple sugar iron (TSI) slants
TSI is a differential medium used to differentiate enterics
based on the ability to reduce sulfur and ferment carbohydrates. If any of the three sugars are fermented, there will be a yellow color change from the acid that is produced. The slant has more oxygen and the butt has less which can influence which organisms grow where. If the organisms form gas as a product of fermentation, there will be cracks or fissures in the agar. Sometimes this manifests as the agar being pushed up from the
bottom of the tube. If an organism can reduce sulfur, the hydrogen sulfide gas which is produced will react with the
iron to form iron sulfide, which appears as a black precipitate. If the precipitate is formed, it can mask any acid/alkaline results. Sulfur reduction requires an acidic environment, so if the black
precipitate is present, some fermentation took place.
Starch Hydrolysis Test
Tests for enzymes that can break down large polysaccharides into smaller molecules that can be used for energy. In our case, a hydrolytic enzyme called amylase and the addition of a water molecule will break the polysaccharide down into usable glucose. Areas of starch hydrolysis will be apparent on the media upon introduction of Gram’s iodine.
Starch Hydrolysis Reagent
Gram’s Iodine
Starch Hydrolysis tests for…
The enzyme amylase
Starch Hydrolysis Positive
A clear halo around the colonies after the addition of Gram’s Iodine.
Starch Hydrolysis Negative
The entire medium is dark blue/brown.
What temp is every test incubated at and for how long?
30°C for 1-3 days.
Which is the only test that uses an inoculation needle instead of the loop?
The motility test, because it uses an agar deep.
Spore Stain Procedure
- Flood the smear with malachite green.
- Place the slide on a staining rack. Pass the Bunsen burner flame under the slide until the stain steams; continue for 5 minutes, replenishing the stain as needed. Do not allow stain to boil or completely evaporate.
- Remove the samples from the heat source and allow the slides to cool.
- Rinse the slides well with water.
- Flood sample with safranin for 60 seconds.
- Rinse well with water.
- Blot dry.
Gram Stain Procedure
- Place stain on stain rack
- Flood slide with crystal violet and let set for about a minute
- Gently rinse with water
- Flood slide with Gram’s Iodine and let set for about a minute
- Pour off Gram’s Iodine
- Add EtOH
- Add a pipette full and let sit for 5-8 seconds only
- Gently rinse with water
- Flood slide with safranin and let set for about a minute
- Gently rinse with water
- Gently blot dry with paper towel
