Microbiology unknown project #40 Write a journal about the identification process. I will add an attached file with all details and requirements. I will also add all results and tests I did. Picture #

Please list your Unknowns culture clearly on your cover page.

Identification of Unknown #6

by

Travis Kibota Biology 240 Winter 2004

This is a journal of activities that I performed on one of the Unknowns cultures during Winter

2004. I identified BOTH organisms in mixed culture #6. You report will contain your work on identifying ONE organism. Note that this example report is not complete.

Biol 240 Winter 2004 Unknowns ID Kibota 2

Journal: Unknown #6 Notice that entries are labeled with the Day 1 (February 3, 2004) Day of observation AND the date.

· Streaked 2 TSA and 2 EMB plates (there were no Azide or MacConkey plates). · Incubated one TSA/one MacConkey at 30°C, other set at 37°C.

Day 2 (February 4, 2004) Observations

· TSA (37°C) –see Figure 1.

On Day 2, I described my observations

of colony characteristics, in detail, for each of the cultures.

o Colony A was ~ 1 mm diameter, entire margin, slightly raised and convex, unpigmented, somewhat translucent.

o Colony B was ~1 mm diameter, entire margin, slightly raised and convex, colored slightly off-white or yellow, more opaque than colony A.

Colony

Colony B Photos are

helpful but not required.

Figure 1. Unknown 6 on TSA incubated at 37°C.

· TSA (37°C)

o Colony A was very similar to 37°C plate.

o Colony B was similar to 30°C plate except colonies were only 1/2 to 2/3 the size.

· EMB (30°C and 37°C)

o The colonies were pinpoint. Because of the small size, I couldn’t reliably determine other characteristics.

Activities

· I picked cells from the colony labeled “A” and streaked onto a TSA plate. I picked cells from the colony labeled “B” and streaked a second TSA plate. I did this to make sure that my cultures are pure. I incubated both plates at 37°C.

· Because my Day 1 plates have only been incubated for 24 hours, I am re-incubating them to encourage the colonies to grow to larger size. Continuing on Day 2, I

described activities I performed AND why I performed them.

Biol 240 Winter 2004 Unknowns ID Kibota 3

Day 3 (February 5, 2004) Observations

· The TSA Plates from Day 1 (both 30°C and 37°C) look very similar to their appearances

on Day 2. The only difference is that Colony B is now ~2-3 mm in diameter. Colony A and B are definitely different colors. Colony A is white (unpigmented). Colony B is more yellowish and may be pigmented (although I’m not sure about this).

· The EMB Plates from Day 1 have grown a little more but the colonies are still small (> 1mm). They appear light colored indicating no lactose fermentation. However, their small size makes this determination difficult.

· The Day 2 TSA Isolation Plates have good growth. Both the Colony A and Colony B plates look homogenous. The colonies look like they do on the Day 1 TSA plates. I am confident I have good isolation (Figure 2A, B).

A B

Figure 2. Day 2 TSA plate with isolated cells from (A) colony A and (B) colony B.

Activities

· I will select cells from one colony on my Colony A plate. I will use these cells to inoculate two TSA slant cultures: one slant will become my working stock, the other will be my reserve.

· I will select cells from one colony on my Colony B plate. I will use these cells to inoculate two TSA slant cultures: one slant will become my working stock, the other will be my reserve.

· I will incubate all four TSA slant cultures at 37°C.

Day 4 (February 6, 2004) Observations

· Colony A Stock and Reserve slants had good homogenous translucent white growth.

· Colony B Stock and Reserve slants had good homogenous opaque yellow growth (Figure 3).

Figure 3. Stock cultures of Colony A (left) and Colony B (right).

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Activities

· Performed Gram-stain for both Colony A and Colony B (using cells from stocks). Both appear to be Gram-positive cocci (Figure 4A, B).

· Refrigerated stock and reserve colonies.

A B

Figure 4. Gram-stains of (A) Colony A and (B) Colony B. Both appear to be Gram-positive cocci.

Day 5 (February 9, 2004) Observations

· None.

Activities

· Given that both colonies appear to be Gram-positive cocci, I ran catalase tests on both cultures. I did this by putting a drop of hydrogen peroxide on a slide (one drop per culture) and mixing cells into the drop. I ran these tests twice for both cultures so I am fairly confident of the results.

Conclusions

· Colony A is catalase NEGATIVE. Thus, I will tentatively conclude that Colony A is either Streptococcus or Enterococcus.

· Colony B is catalase POSITIVE. Thus, I will tentatively conclude that Colony B is either Staphylococcus or Micrococcus. At this point, I have begun to narrow down the

possibilities. Notice that I listed the possible organisms. Here I listed only the genus names.

Day 6 (February 10, 2004) This indicates that I can have any of the species Observations within those genera.

· None.

Activities

· Because the catalase result is so critical to my next steps, I want better confirmation. On a TSA plate, I heavily inoculated Colony A on the left side and Colony B on the right

Biol 240 Winter 2004 Unknowns ID Kibota 5

side. After incubation, I will put drops of hydrogen peroxide on each area of growth. This will provide a much more sensitive test for catalase ability.

· I streaked Colony A and Colony B on blood agar plates to test for hemolytic ability. · I would like to test Colony B on 7% NaCl, but no plates are available.

Day 7 (February 11, 2004) Observations

· TSA had heavy growth of both Colony A and Colony B (Figure 5). This plate shows the difference in pigmentation. I am now confident that Colony A is unpigmented and Colony B produces a yellow, water-insoluble pigment.

Hydrogen peroxide with no bubbles

Large, vigorous bubbles

Figure 5. This shows a TSA plate that has heavy growth of Colony A and Colony B. Hydrogen peroxide was dropped on both regions of growth. No bubbles appeared in the Colony A region. Vigorous bubbling appeared in the Colony B region.

· Colony A showed no hemolysis on blood agar.

· Colony B showed complete (!) hemolysis on blood agar (Figure 6).

A B

Figure 6. Blood agar plates. (A) Colony A formed small white colonies that show no hemolysis. (B) Colony B formed small white colonies that show complete (!) hemolysis.

Biol 240 Winter 2004 Unknowns ID Kibota 6

Summary of observations

Characteristic Gram-Reaction

Cell Shape Pigment Catalase Hemolysis Possible Species

Here I present a table of characteristics and possible organisms based on these characteristics. Notice that I’ve now begun to list individual species (instead of just the genus names) because I’m narrowing down the possibilities.

Colony A Gram-positive Coccus

Negative Negative Negative

Streptococcus pneumoniae Streptococcus mitis Streptococcus bovis Enterococcus faecalis

!-hemolyzers (tentatively ruled out)

Streptococcus pyogenes Streptococcus agalactiae

Colony B Gram-positive Coccus

Yellow Positive Beta

Staphylococcus aureus

Micrococcus luteus is pigmented yellow but not beta-hemolytic.

Staphylococcus epidermidis in unpigmented and not beta-hemolytic.

Activities

· For Colony A, I inoculated a Bile Esculin slant. This will separate E. faecalis/S. bovis from S. mitis/S. pneumoniae.

· For Colony B, I am fairly confident that it is Staphylococcus aureus. I inoculated a mannitol broth. If positive for mannitol fermentation, this will add to my confidence. When 7% NaCl becomes available, I will test this (to defintively rule out Micrococcus).

Day 8 (February 12, 2004) Observations

· Colony A Bile Esculin: The culture turned black from top to bottom. Bile esculin is considered positive if more than half the culture is black, so Colony A is definitely positive (Figure 7).

· Colony B Mannitol Broth: The broth turned bright yellow with no gas bubble. Thus, Colony B is an acid-former in Mannitol (Figure 8).

Summary of Observations

Biol 240 Winter 2004 Unknowns ID Kibota 7

Figure 7. Bile Esculin Test. Control (left) and Colony A (right). Note the dark coloration of the Colony A tube relative to the control.

Figure 8. Mannitol Broth. Control (left) and Colony B (right). Colony B shows acid production (yellow) but no gas bubble.

Conclusions/Activities

· Colony A is narrowed down to Streptococcus bovis or Enterococcus faecalis. To differentiate between these two species, I inoculated a 6.5% NaCl Broth—E. faecalis should be positive for growth, S. bovis should be negative.

· I am fairly confident that Colony B is Staphylococcus aureus. However, Micrococcus luteus is currently only ruled out by hemolytic ability. To increase confidence, I inoculated a 7% NaCl plate—S. aureus should be positive for growth, M. luteus should not grow.

I didn’t finish these identifications. If I did, I would draw final conclusions about my organisms. I would present these conclusions in a separate section that would clearly list: the Unknowns Number and the organisms that I think I have.

Following this conclusion section, I would provide a brief (one paragraph) write up about my organisms. This write-up would be based on literature research about my organisms. I would include literature citations for this research.