Microbiology Bacterial Unknown Project

MICROBIOLOGY BACTERIAL UNKNOWN

PROJECT UNKNOWN BACTERIAL CULTURE #18

BY: MADISON PARKER

TEMPERATURE OPTIMUM • The lower the % transmittance

the more turbid the broth. This indicates more growth at that specific temperature. The optimum temperature to growth the unknown bacterial culture was at 30C because at that temperature, the % transmittance was lowest.

Temperature % Transmittance

25C 48%

30C 30%

37C 43.5%

AGAR SLANT CHARACTERISTICS • Moderate to large

abundance of growth• Nonchromogenic-

white/gray colonies• Opaque or similar

appearance to “frosted glass”

• Echinulate form-continuous, thread-like with irregular edges

COLONY CHARACTERISTICS

• Moderate to large size• White or gray

colonies, opaque• Irregular form• Undulate/Lobate

margins • Raised-slightly

elevated

BROTH GROWTH PATTERNS

• Uniform fine turbidity and finely dispersed throughout

• Soft, easily dispersed sediment

GELATIN

• 1st time performed=liquefaction

• 2nd time performed slightly liquefied

• Liquefaction =hydrolysis of gelatin

SIMPLE POSITIVE STAIN MORPHOLOGY

• The simple stain helped make it easier to see the cell by increasing the contrast between the cell and it’s background.

• The unknown was found to have a rod or bacillus form.

NEGATIVE STAIN MORPHOLOGY

• The negative stain helped to confirm that the unknown bacteria did in fact have a rod/bacillus form.

CELL MEASUREMENT • The oil-immersion lens was used to

measure the size of each bacteria cell.

• The cell was measured at 3 ocular divisions by 1 ocular division.

• The conversion factor for the oil-immersion lens was 1μm/ocular division.

• Therefore, the cell measurements were:

Length=3μm

Width=1μm

GRAM STAIN

• The gram stain showed the cells stained purple. This indicated that the cells retained the crystal violet stain and are therefore classified as gram-positive.

ACID FAST STAIN

• The unknown bacteria picked up the counterstain methylene blue which indicated that the bacteria was non-acid fast.

• This indicated that the bacteria did not belong to the genera Mycobacterium or Nocardia

• This was a negative test

CAPSULE STAIN

• Small white halos were not observed around the unknown bacillus. The white circles were determined to be bubbles in the stain. The stain performed indicated that the unknown bacteria did not utilize capsules.


ENDOSPORE STAIN

• There were endospores present within the unknown bacteria.

• The green malachite green stain remained in the endospores and the cytoplasm of the vegetative cells were counterstained with safranin.

• This was a positive test

PHB STAIN • Sudan Black B Stain was utilized for this

stain. If granules were present in the unknown, the Sudan Black B would have stained the poly-B-hydroxybutyric acid granules dark blue.

• The technique we used to stain the granules dark blue and cytoplasm red.

• The unknown bacterium did not have blue black granules that were present after the staining and therefore does not have PHB granules.


GAS PAK OXYGEN REQUIREMENTS• The unknown bacteria had

similar growth to E. Coli bacteria.

• The unknown was able to grow in the presence and absence of oxygen.

• E. Coli is a facultative anaerobe. • These results are consistent

with facultative anaerobic growth.

THIOGLYCOLLATE BROTH• The unknown thioglycollate

tube showed growth in the top 3/4ths of the tube.

• Aerobes grow near the top of the broth and facultative species grow throughout the tube.

• These results are consistent with facultative species.

• This test result was consistent with the gas pak test.

CANDLE JAR TEST • Since there was not a candle jar at 30C,

the unknown was growth at the second best temperature, 37C.

• In the candle jar test, the unknown bacteria grew similarly to the E. Coli control. The unknown bacteria was able to grow under aerobic and anaerobic conditions.

• These results are consistent with bacteria that are facultative anerobes.

• These test results were consistent with the gas pak and thioglycollate broth tube test.

OSMOTIC EFFECTS • The bacteria appeared as white and

yellow colonies with the most growth on agar that contained 0% and 5% of NaCl.

• As the NaCl% increased, the colony color decreased and there was less growth. Agar that contained 10% and 15% had some growth present but very little.

• These results showed the bacteria could not survive in environments with high salt content present.

% NaCl Growth Pigment

0% Most Growth White/yellow

5% 3+ White

7.5% 2+ White

10% Some growth Specs growth

15% Some growth Specs of growth

STARCH HYDROLYSIS

• There was no clearing around the bacteria.

• The presence of the blue/black coloring after the addition of iodine indicated that the unknown bacteria could not hydrolyze starch.


PROTEIN (MILK) HYDROLYSIS • The bacteria grew on the milk

agar and there was a wide zone of hydrolysis present.

• This indicated that the unknown bacteria could hydrolyze the protein casein in milk and use it as an energy source.


FAT HYDROLYSIS • In this test, the unknown

bacteria was able to grow on the media; however, there was an absence of the zone of hydrolysis.

• This indicated that the unknown bacteria could not hydrolyze fat as an energy source.


DNA HYDROLYSIS • In this test, it looked as if there was a

zone of hydrolysis around the bacteria growth but it was very hard to see.

• The picture on the right was taken after HCl was added to the agar.

• The addition of 0.1 M of HCl polymerizes the intact DNA and made it more obvious to see.

• This test indicates the hydrolysis of DNA due to the clearing of methyl green around the colony.


LACTOSE FERMENTATION • The lactose fermentation broth results

showed an acid byproduct produced. This was indicated by the color change of the methyl red from red to yellow.

• The results also showed gas production, indicated by the gas trapped in the Durham tube, which displaces the medium.

• These results suggest that the unknown bacteria can ferment lactose


SUCROSE FERMENTATION • Due to the lack of color change by

the methyl red indicator, this test shows that there were no acid byproducts produced.

• There was also no gas production indicated by the Durham tube.

• These test results indicate that the unknown bacteria can not ferment sucrose


MALTOSE FERMENTATION • The maltose fermentation broth results



• These results show that the unknown bacteria can ferment maltose


GLUCOSE FERMENTATION

• The glucose fermentation broth results showed an acid byproduct produced. This was indicated by the color change of the methyl red from red to yellow.


• These results show that the unknown bacteria can ferment glucose.


MANNOSE FERMENTATION • The mannose fermentation broth results



• These results showed that the unknown bacteria can ferment mannose.


ONPG • A positive ONPG test is the

appearance of yellow color in the test tube.

• This indicates that the ONPG test is negative.

• Because the lactose test was positive and this test is negative, that means that the unknown bacteria


TRIPLE SUGAR-IRON AGAR TEST-24HRS

• After 24hs the TSI tube showed yellow throughout the tube which indicated that fermentation of the sugars within the agar were being fermented and utilized as an energy source.

• This test indicated fermentative metabolism due to the acid production in the butt of the tube after 24 hours.

TRIPLE SUGAR-IRON AGAR TEST-48HRS+

• After 48hrs the tube had a yellow butt and a red slant.

• This is also indicated of fermentative metabolism.

• This shows that only glucose fermentation occurred, then the organism was unable to ferment the lactose or sucrose and so it switch over to deaminating proteins.

• Due to previous tests, it is known that the unknown bacteria cannot ferment sucrose.

• It can be assumed that the unknown bacteria fermented the glucose and lactose within the first 24 hours and then switched over to deaminate proteins as an energy source because it was unable to ferment the sucrose present in the agar.

LITMUS MILK REACTIONS-48HRS

• The first observation of the litmus milk reactions showed a pink color starting to develop and a precipitate beginning to form at the bottom of the test tube.

• Because multiple reaction become apparent at different time intervals, the litmus milk reactions are observed at 48hrs and 5 days.

LITMUS MILK REACTIONS-5 DAYS • The pink on top is due to the utilization of lactose. If acid is

produced, they cause a shift in pH to the acidic range which is indicated in the change in the litmus indicator from purple to pink. These results are consistent with the lactose fermentation tube results.

• Curd was observed at the bottom of the test tube. It was determined that it was acid curd which occurs when the pH of the medium decreases to 5 or lower. When the curd was analyzed it was hard which is consistent with the acid curd description.

• Peponization occurs when the protein is digested. It is indicated by a clearing in medium that begins to appear at the topof the tube and proceeds downward. In addition, the medium takes a brownish color.

• These results indicated acid byproducts, followed by reduction and the formation of acid curd.

OXIDASE TEST • The unknown bacteria was tested to determine if

cytochrome oxidase was present. This is an enzyme that is present in the ETS of some bacteria.

• The unknown bacteria was tested on the right side. The blue color appeared within 30s.

• This indicated the presence of cytochrome oxidase.

• These test results indicate that the unknown bacteria can utilize aerobic respiration.

• These results are consistent with the previous gas pak, candle jar, and thioglycollate tests in which the unknown bacteria was found to be able to growth in the presence of oxygen and the absence of oxygen.


CATALASE TEST

• After hydrogen peroxide was added to the slide, bubbles were observed.

• This indicated that the unknown bacteria can detoxify H2O2.


NITRATE REDUCTION • This test helped to determine if the unknown bacteria

could reduce nitrate.

• The results did not indicate the formation of gas, however there was heavy growth on the bottom of the test tube.

• After the sulfonic acid and methyl-a-naphthalene was added, the test tube turned red which is indicative of a positive test.

• These results show that nitrate was reduced to nitrite and that anaerobic respiration has taken place.

• E. Coli has similar results in that it only reduces the nitrate to nitrite. The unknown bacteria and E. Coli have similar growth conditions in being facultative anaerobes. This means they can utilize oxygen or another inorganic terminal electron acceptor. The nitrate reduction results are consistent with previous tests performed.


INDOLE TEST • In this test, the ability of the unknown bacteria to degrade the amino acid tryptophan was tested.

• A positive test is indicated by the development of a rose color after Kovac’s reagent was added to the test tube.

• The tube did not undergo a color change. This indicates a negative test and the unknown bacteria could not hydrolyze tryptophan.


METHYL RED • The goal of the methyl red test is to

detect the presence of large concentrations of acid end products.

• When large concentrations of acid byproducts are produced and the pH of the media is lower below 4.4, the pH indicator changes from yellow to red.

• This test indicates that the unknown bacteria switches to mixed acid type glucose fermentation and produces large quantities of acid byproducts.


VOGES-PROSKAUER • The Voges-Proskauer test indicates the

formation of nonacidic or neutral byproducts which occurs during the butylene glycol type glucose fermentation.

• Because the test tube did not turn a rose color when alpha mapthol and KOH+ creatine was added, this indicates that the unknown bacteria does not utilize the butylene glycol type glucose fermentation and it does not produce nonacidic or neutral byproducts.


CITRATE • The results showed that there was

no color change, some growth on the slant surface; however, there was also growth in the stab inoculation.

• The absence of color change from green to blue means this was a negative test and the unknown bacteria can not utilize citrate as a carbon source.


PHENYLALANINE

• There was no green color change that occurred after incubation

• This test indicated that the unknown bacteria cannot deaminate phenylalanine and utilized by the unknown bacteria.


UREA• There was no growth on the

urea agar or color change from orange to bright pink.

• These test results indicated that the unknown bacteria does not produce the enzyme urease, which splits urea into ammonium and carbon dioxide.


LYSINE

• Due to the absence of purple color, this test indicates that the unknown bacteria cannot decarboxylate lysine and utilize it to neutralize environments.


ORNITHINE-24HRS

• The color change from clear to blue/purple indicated a positive test.

• This means that the unknown bacteria is able to decarboxylate ornithine in order to neutralize its acidic environment.


ORNITHINE-48HRS • The results after 48hrs continued to show a positive test.

• When microorganisms ferment, they often produce acidic waste products that make the medium inhospitable.

• Previously, the unknown bacteria was found to utilize mixed acid type glucose fermentation, in which the unknown produces a large concentration of acid byproducts. These results are consistent with that test in that the unknown would need to be able to decarboxylate specific amino acids in order to neutralize it’s environment.

ARGININE • The results did not show a color

change from orange to dark rose. There was also no growth.

• The tube on the left is an un-inoculated arginine tube, the tube in the middle is the unknown bacteria, and the tube on the right is a positive test. This shows that the unknown bacteria had a negative test and that it is unable to deaminate arginine.


MOTILITY AGAR • This agar had lower concentration of

agar (0.5%) than a normal agar plate. This gives it a semi-solid consistency and allows for motile bacteria to be tested for.

• The diffuse growth that spread from the area where the agar was stabbed indicates that the unknown bacteria is motile.

• A hanging drop was also performed in order to determine if the unknown bacteria was motile. The hanging drop also showed that the unknown bacteria was motile.


MANNITOL SALT AGAR • There was no growth present on the mannitol salt agar.

• Manitol salt agar contains high salt content and also contains the pH indicator phenol red and mannitol sugar to determine if the bacteria can ferment mannitol.

• The lack of growth was due to the high salt concentration. The unknown bacteria is unable to grow at high salt concentrations. This is consistent with the NaCl test performed previously.


BLOOD AGAR • The unknown bacteria did not

did not break down the red blood cells on the blood agar and there was no amount of clearing present.

• These results show that the unknown bacteria has beta hemolysis.

• This was a positive test for hemolysis

BACILLUS CEREUS

UNKNOWN BACTERIA #18

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Microbiology Bacterial Unknown Project