Microbiology Practical #2

Front 1

Four quadrant streak plate method

Back 1

1) Using aseptic technique, streak one loopful of the organism in one small section of the plate (hugging the edge) making 3 - 4 zigzags. Cover the plate.
2) Flame the loop and touch to the uninoculated corner of the first quadrant area to cool the loop. Make 5 - 6 zigzags starting in the first quadrant and dragging organism into the second. Cover the plate.
3) Flame the loop and touch to the uninoculated corner of the first quadrant area. Make 5 - 6 zigzags dragging the organism from the second quadrant into the third. Cover the plate.
4) Flame the loop and touch to the uninoculated corner of the first quadrant area. Make 5 - 6 zigzags dragging the organism from the third quadrant into the fourth. Make certain not to connect the fourth area back into the first. Cover the plate.

Front 2

Pour plate method

Back 2

(The advantage to using this method is that it requires less skill than the streak plate method. However, it is important to note that it does require three times more media.):
1) Liquefy 3 tubes of nutrient agar (label 1, 2 and 3) by boiling in water bath for 5 minutes. Label three empty, sterile Petri plates 1, 2 & 3.
2) Cool water bath to 50oC and let stand for 5 minutes.
3) Disperse the organism in its tube by rotating gently.
4) Transfer one loopful of the broth culture into tube 1.
5) Disperse the organism in tube 1.
6) Transfer one loopful of the agar from tube 1 to tube 2.
7) Disperse the organism in tube 2.
8) Transfer one loopful of the agar from tube 2 to tube 3.
9) Disperse the organism in tube 3.
10) Pour the inoculated agar from each tube into its respective plate. Swirl to distribute agar and organism evenly over the bottom of the plate. Place lid slightly ajar to allow steam condensation out of plate. After the agar has completely solidified, invert and incubate plates.

Front 3

Why

Back 3

to achieve a pure culture by creating isolated colonies

Front 4

Culture Media Preperation
Nutrient Agar:

Back 4

Measure the desired amount of dehydrated powder according to the package directions. Add the powder to de-ionized or distilled water (tap water inhibits the growth of bacteria). Bring into solution by boiling for 1 minute while stirring. Dispense to test tubes etc. Label with media name and date, cap with sponge and sterilize in preheated autoclave on liquid cycle (121oC @ 15 PSI for 15 minutes). The pressure inside the autoclave is exhausted slowly to avoid breaking glass tubes or boiling over agar. Slants can be formed by tilting rack after media is removed from the autoclave but before it has cooled and solidified.

Front 5

Culture Media Preperation

Nutrient Broth:

Back 5

Same as agar except: 1) do not heat; 2) cap with screw caps loosely to allow gas pressure exchange during autoclaving.

Front 6

Cultivation of Anaerobes:

Gas Pak

Back 6

The Gas-Pak is a strictly anaerobic environment. Hydrogen is generated in the jar, which removes oxygen by forming water. Palladium tablets, in the screen wire basket, catalyze this reaction so that it can occur at room temperature. Water is added to the foil envelope containing tablets which generate hydrogen and carbon dioxide. The methylene blue strip is used as an indicator. When first opened it is colorless because it is sealed in an anaerobic foil pouch, but it quickly turns blue after exposure to atmospheric oxygen. If the strip does not turn back to colorless within two hours, then an anaerobic environment was not created.

Front 7

Cultivation of Anaerobes:

FTM (Fluid Thioglycollate Media)

Back 7

Be sure to determine that the top pink layer is 30% of the overall the volume or less, before inoculating a fresh tube of FTM by stabbing to bottom with a needle. The pink layer indicates the area where oxygen is present. When evaluating tubes of FTM (Fluid Thioglycollate Media) it is important not to agitate the tubes because the position of growth in the medium can be easily changed if handled carelessly.

Front 8

After incubation, evaluate the results of FTM (Fluid Thioglycollate Media) as follows:

Back 8

AEROBIC - growth is only on the top of the medium (the pink area) which indicates the bacteria cannot exist without oxygen
MICROAEROPHILIC - growth is only in the bottom half of the formerly pink area, which indicates the bacteria, use oxygen but only in limited amounts
FACULTATIVE - growth is throughout the medium which indicates that the bacteria has enzyme systems enabling them to utilize free oxygen or some alternative oxygen source such as nitrate (if oxygen is present, they tend to use it in preference to the alternatives).
ANAEROBIC - growth has developed on the bottom which indicates that the bacteria requires an oxygen-free environment and would die in the presence of oxygen

Front 9

Morphological study of unknown bacterium: Know the steps necessary to make pure culture reserve and working stock cultures.

Back 9

Inoculate with a needle in a straight streak up the surface only of 2 nutrient agar slants from the middle of an isolated colony from a streak plate. Incubate one slant at body temperature (37oC) and one slant at room temperature (24oC) for 24 - 48 hours. Gram stain both to check for purity.

Reserve and working stock cultures are started from an isolated colony (one that is uniformly margined and not touching any other colony or the edge of the plate). Additionally, the best growing slant is transferred after incubation to the refrigerator as Reserve. It will only be used to inoculate another working stock slant as needed. The Working stock culture is also transferred to the refrigerator and used for making routine inoculations and slides. Fresh working should be made every 10 days to two weeks from the Reserve

Front 10

Know how to read motility stab.

Back 10

Since the culture tube method of motility determination is recommended with a pathogenic organism, this is also the method of choice with an Unknown that may be pathogenic (better safe than sorry). Cloudiness indicates motility. Growth only along the stab line indicates non-motility.

Front 11

Four quadrant streak plate method

Back 11

1) Using aseptic technique, streak one loopful of the organism in one small section of the plate (hugging the edge) making 3 - 4 zigzags. Cover the plate.
2) Flame the loop and touch to the uninoculated corner of the first quadrant area to cool the loop. Make 5 - 6 zigzags starting in the first quadrant and dragging organism into the second. Cover the plate.
3) Flame the loop and touch to the uninoculated corner of the first quadrant area. Make 5 - 6 zigzags dragging the organism from the second quadrant into the third. Cover the plate.
4) Flame the loop and touch to the uninoculated corner of the first quadrant area. Make 5 - 6 zigzags dragging the organism from the third quadrant into the fourth. Make certain not to connect the fourth area back into the first. Cover the plate.

Front 12

Pour plate method

Back 12

(The advantage to using this method is that it requires less skill than the streak plate method. However, it is important to note that it does require three times more media.):
1) Liquefy 3 tubes of nutrient agar (label 1, 2 and 3) by boiling in water bath for 5 minutes. Label three empty, sterile Petri plates 1, 2 & 3.
2) Cool water bath to 50oC and let stand for 5 minutes.
3) Disperse the organism in its tube by rotating gently.
4) Transfer one loopful of the broth culture into tube 1.
5) Disperse the organism in tube 1.
6) Transfer one loopful of the agar from tube 1 to tube 2.
7) Disperse the organism in tube 2.
8) Transfer one loopful of the agar from tube 2 to tube 3.
9) Disperse the organism in tube 3.
10) Pour the inoculated agar from each tube into its respective plate. Swirl to distribute agar and organism evenly over the bottom of the plate. Place lid slightly ajar to allow steam condensation out of plate. After the agar has completely solidified, invert and incubate plates.

Front 13

Why

Back 13

to achieve a pure culture by creating isolated colonies

Front 14

Culture Media Preperation
Nutrient Agar:

Back 14

Measure the desired amount of dehydrated powder according to the package directions. Add the powder to de-ionized or distilled water (tap water inhibits the growth of bacteria). Bring into solution by boiling for 1 minute while stirring. Dispense to test tubes etc. Label with media name and date, cap with sponge and sterilize in preheated autoclave on liquid cycle (121oC @ 15 PSI for 15 minutes). The pressure inside the autoclave is exhausted slowly to avoid breaking glass tubes or boiling over agar. Slants can be formed by tilting rack after media is removed from the autoclave but before it has cooled and solidified.

Front 15

Culture Media Preperation

Nutrient Broth:

Back 15

Same as agar except: 1) do not heat; 2) cap with screw caps loosely to allow gas pressure exchange during autoclaving.

Front 16

When should you do an acid fast stain.

Back 16

a Gram positive rod that is not a spore former should be tested for acid-fastness. Although some bacteria require four to five days growth to exhibit acid-fastness, most become so, at least partially, within two days. Still it is important not to use cultures that are too old. As most bacteria do not produce cells that are 100% acid-fast, an organism is considered acid-fast even if only portions of the cells exhibit this characteristic.

Front 17

When should you do a spore stain.

Back 17

If an Unknown is a Gram positive rod, a spore stain should be done. It is important to note; however, that spore formers can require additional incubation before sporalation occurs so it is prudent to recheck a negative result after additional incubation. Rarely is a coccus or Gram negative rod a spore former

Front 18

Know Gram reactions

Back 18

Purple - Gram positive

Pink - Gram negative

Front 19

Cultural Characteristics

Know how to read gelatin liquefaction.

Back 19

Nutrient gelatin is used to test for gelatin hydrolysis or liquefaction. Organisms that are able to liquefy gelatin produce the enzyme gelatinase. Inoculate with a straight stab to the bottom of the media using a needle. Incubate for at least 24 hours but it will more likely take closer to 30 days. After incubation, place in refrigerator or ice bath for at least five minutes. Tilt the COLD tube to see if any portion of the medium is still liquid. If it is liquid while cold, record the positive reaction for gelatin liquefaction. If it is completely solidified, return it to the incubator to be checked occasionally (by returning to the ice bath or refrigerator for at least thirty minutes) over the next 30 days or until it shows positive.

Front 20

Oxidation and Fermentation Reactions:

Phenol Red Glucose, Lactose & Mannitol broth

Back 20

Media contains: Phenol Red and appropriate carbohydrate
Tests for fermentation of selected carbohydrate.
+ (for acid production from fermentation) if yellow
- (for acid production from fermentation) if red
+ (for gas production from fermentation) if bubbles in Durham tube
- (for gas production from fermentation) if no bubble in Durham tube

Front 21

Oxidation and Fermentation Reactions:

MR-VP

Back 21

Media contains: glucose
Tests for mixed acids formed as a result of glucose fermentation if the enzyme formic hydrogenase is present to split the formic acid yielded during glucose fermentation into CO2 and H2
After adding 3 - 4 drops of the methyl red reagent...
+ (Methyl Red test) if red which indicates large amounts of acids
- (Methyl Red test) if yellow which indicates small amounts of acids

Tests for production of acetoin & 2,3 butanediol from glucose fermentation After adding a dropperful each of Barritt’s reagents A & B and shaking for up to two hours...
+ (Voges-Proskauer test) if red which indicates acetoin is present
- (Voges-Proskauer test) if brown which indicates acetoin is absent

Front 22

Oxidase and Fermentation Reactions:
Tests for catalase production, which breaks down hydrogen peroxide to form water and oxygen

Back 22

After adding 3 drops of hydrogen peroxide to the organism on a slant, plate or just a slide (no special media is required)...
+ (Catalase Production test) if bubbles form on organism
- (Catalase Production test) if no bubbles form on organism

Front 23

Tests for oxidase production

Back 23

After adding disk saturated in oxidase reagent and a drop of sterile water to the organism on a plate, slant or just a slide (no special media is required)...
+ (Oxidase Production test) if colonies turn pink, to red, to black
- (Oxidase Production test) if colonies remain the same

Front 24

Oxidation and Fermentation Reactions:

Nitrate Agar Slant

Back 24

Media contains: potassium nitrate
Tests for nitratase which reduces nitrate to nitrite and / or nitrogen
After adding 3 drops each of Nitrite reagents A & B ...
+ (Nitrate Reduction test) if media turns red
- (Nitrate Reduction test) if media remains clear
Negatives should be confirmed by adding a pinch of zinc dust ...
+ (Nitrate Reduction test) if media still remains clear
- (Nitrate Reduction test) if media only turns red after adding zinc

Front 25

Hydrolytic Tests

Back 25

Media contains: starch
Tests for hydrolysis of starch, which requires amylase, to yield maltose, glucose and dextrins
After adding Gram’s iodine over the growth to darken the starch containing media...
+ (Starch Hydrolysis test) if clear zone appears around the growth
- (Starch Hydrolysis test) if no clear zone appears around the growth

Front 26

Hydrolytic Tests

Skim Milk Agar Plate

Back 26

Media contains: milk protein - casein
Tests for hydrolysis of casein (peptonization) to produce transparent derivatives because of the exoenzyme caseinase
+ (Casein Hydrolysis test) if clear zone exists around the growth
- (Casein Hydrolysis test) if the media is opaque under and around the growth

Front 27

Hydrolytic Tests

Spirit Blue Agar Plate

Back 27

Media contains: vegetable oil
Tests for hydrolysis of fat with the lipase enzyme to form a molecule of glycerol and three fatty acid molecules which usually lowers the pH
+ (Fat Hydrolysis test) if dark blue precipitate forms on the growth or the oil droplets disappear around the growth
- (Fat Hydrolysis test) if no precipitate forms and droplets remain

Front 28

Hydrolytic Tests

Tryptone Broth

Back 28

Media contains: tryptophan
Tests for hydrolysis of amino acid tryptophan requiring the enzyme tryptophanase to form indole and pyruvic acid
After adding 10 to 12 drops of the Kovac’s reagent
+ (Indole Production test) if a red ring or layer of reagent forms on top
- (Indole Production test) if yellow ring or layer forms

Front 29

Hydrolytic Tests

Urea Broth

Back 29

Media contains: urea and phenol red indicator
Tests for urea hydrolysis, which yields ammonia to raise the pH if the urease enzyme is present
+ (Urea Hydrolysis test) if media become pink (cerise) or red
- (Urea Hydrolysis test) if media remains orange -
Should be reincubated if negative

Front 30

Biochemical Tests

Kligler’s Iron Agar Slant

Back 30

Media contains: iron salts, glucose, lactose and a phenol red indicator
Tests for hydrogen sulfide production from cysteine due to the enzyme cysteine desulferase as well as glucose and lactose fermentation
+ (H2S Production test) if black precipitate of iron sulfide forms in stab at bottom of tube
- (H2S Production test) if no black precipitate forms in stab at bottom of tube
+ (Glucose Fermentation test) if yellow color forms at bottom of tube
- (Glucose Fermentation test) if red color forms at bottom of tube
+ (Lactose Fermentation test) if yellow color forms in slant of tube
- (Lactose fermentation test) if red color forms in slant of tube

Front 31

Biochemical Tests

Simmon’s Citrate Agar Slant

Back 31

Media contains: citrate as sole carbon source
Tests for ability of organism to utilize citrate as sole carbon source
+ (Citrate Utilization test) if slant turns blue and organism grows
- (Citrate Utilization test) if slant stays green and shows no growth

Front 32

Biochemical Tests

Phenylalanine Agar Slant

Back 32

Media contains: phenylalanine
Tests for deamination of the amino acid phenylalanine using the enzyme phenylalanase to produce phenylpyruvic acid and ammonia
After adding 5 - 10 drops of the ferric chloride reagent...
+ (Phenylalanine Deamination test) if green color forms on surface of slant
- (Phenylalanine Deamination test) if no green color forms on slant

Front 33

Biochemical Tests

Litmus Milk

Back 33

Media contains: milk and litmus indicator
Tests for reactions on milk
Acid (Litmus Milk Reaction) if media turns pink
Alkaline (Litmus Milk Reaction) if media is purple
Reduction (Litmus Milk Reaction) if media become colorless
Coagulation (Litmus Milk Reaction) if media becomes solid
Peptonization (Litmus Milk Reaction) if media becomes translucent