Fungi

Rhizopus stolonifer

Aspergillus niger

Penicillium notatum/chrysogenum

Class Room Activities

Collecting our own saliva, yum

Flame that loop!Jodi carefully labels a specimen

To Know and be Known

We started out making working slants for our unknown. Next we isolated our unknowns on PEA, MSA and EMB. My #30 made the same flourescent yellow halo on MSA as the control Staph. aureus did. It also would show similare growth and fermentation patterns when compared to Staph. aureus as a control. In this way I was lucky, since most of the other unknowns did not use a control that was the same microbe. Our controls where usually S. aureus, Bacillus subtilis, E. coli, Psuedomonas aerugenosa or Proteus vulgaris. Still from the MSA experiment onward I was pretty sure I was dealing with S. aureus and it soon became my favorite microbe. It's a very common microbe that lives as normal flora on the skin and nasal passages. It can become a pathogen if there is a break in the skin and the microbe enteres the sterile tissue below. Infact I saw a show about a girl who obtained a S. aureus infection in her brain due to a head injury. There is a highly virulent strain called MRSA S. aures. MRSA stands for Methicillin Resistant Staph. Aureus. My lab partner was shown to have this after we did nasal swabs and isolted our own microbes. She was put on some heavy antibiotics to clear herself of carriage. I hope I didn't pick up this hand me down from her. Staph aureus is a non-spore forming cocci that forms irregulare groups or clumps. The name Satphylococcus Aureus in in Greek literally means golden clusters of grapes. When isolated on media it almost always produces a golden colored growth. It is non-motile and a facultative aerobe which means it prefers aerobic conditions but will also grow, though not as well, in anaerobic conditions. Staph. aureus is Catalase positive and Oxidase negative. It test positive for Gelatinase, DNase and Lypase but does not posses the enzyme amylase. Staph. aureus tolerates NaCl and ferments mannitol. It also tolerates phenyl ethyl alcohol and Eosin-methyline blue. It is Urease negative and MR+ and VP +/-. The IMViC test for Staph. aureus will be completely negative and ferments Sucrose, Lactose or Glucose. My unknown also had similare growth patterns to the S. aureus control when comparing resistance and susceptability to Iodine, Alcohol, H2O2 and Lysol. Just like Staph. aureus it was Susceptable to H2O2, Lysol and Iodine but resistant to Alcohol. This wasn't surprising since S. aureus will grow on Phenyl Ethyl Alcohol agar. Yellow growth on my working slant as well as growth in nutrient broth.

Golden colonies on Nutrient Agar

Similare growth on MSA to Staph. aureus. Tolerates NaCl and ferments Mannitol.

Similare growth on EMB to Staph. aureus. Tolerates Eosin but does not ferment.

Similare growth on PEA to Staph. aureus. Tolerates PEA.

Urease -, MR-VP - (MR should be +), Citrate -, SIM -

Media indicates #30 is facultative

#30 shows similare resistance to H2O2, Lysol and Iodine as Staph. aureus and Resistance to Alcohol

#30 golden growth and starch hydolysis

Ferments Sucrose, Lactose and Glucose
DNase +

Lipase + (slight halo)

Golden growth on Starch Agar

Does not posses enzyme Amylase

4 Unknown Microbes

A. Non-pigmented (white) colonies on BAP. Gamma hemolysis on blood agar. Poor growth and does not ferment Mannitol. Tolersates bile and hydrolyzes esculin. Groth in 6.5% NaCl broth @ 42c. DNase negative. "S" Novobiocin "S" SXT, though this maybe an error on my part "R" Bacitracin "R" Optochin.
Organism most likely Enterococcus faecalis

B. Pigmented (yellow) colonies on BAP. Beta hemolysis on on BAP. Tolerates NaCl and ferments Mannitol. Tolerates bile but does not hydrolyze esculin. Growth in 6.5% NaCl broth @ 42c. DNase positive. "S" Novobiocin "S" SXT "R" Bacitracin "R" Optochin.
Organism is most likely Staphylococcus aureus

C. Non-pigmented colonies on BAP. Gamma hemolysis on BAP. Tolerates NaCl but does not ferment Mannitol. Tolerates Bile but does not hydrolyze esculin. Growth in 6.5% NaCl broth @42c. DNase positive. "S" Novobiocin "S" SXT "R" Bacitracin "R" Optochin.
Organism is most likely Staphylococcus epidermidis

E. Don't ask where D went cuz I don't know.
Non-pigmented colonies on BAP. Beta hemolysis on BAP. Does not tolerate NaCl and does not ferment Mannitol. Does not tolerate bile and does not hydrolyze esculin. No growth in 6.5% NaCl broth @ 42c. DNase positive. "S" Novobiocin "R" SXT "S" Bacitracin "R" Optochin.
Organism is most likely Streptococcus pyogenes

This Weeks Media

I tested my unknown in a variety of media.

Here are the results:

Urea -

Gelatinase error in media

Citrate -

24 Tube Inoculating

We inoculated tubes containing sucrose, glucose, lactose or nutrient gelatin. The liquid tubes are to test carbohydrate utilization. These tubes also conatined a durham tube which is used to indicate gas production. The tubes all had Phenol-red as the ph indicator. This procedure is done to determine if a microbe is facultative, deriving it's energy from fermentation. The fermentation will result in acid production which will lower the pH of the broth and the broth will turn yellow. If not full yellow, the result is negative. Some organisms will also produce amonia and CO2. The durham tube will capture this any gas produced.
The gelatin tubes are to show gelatin hydolysis from the enzyme gelatinase. The protein gelatin is digested by some organisms. This test determines if the microbe does or does not produce gelatinase. If the organism does, the gelatin will change from solid to liquid.
We also made a Qudrant streak plate on Starch Agar and Tributyrin Agar streaked with E. coli, Bacillus subtilis and our unknown. Starch agar selects organisms that produce the enzyme amylase to break down starch. Starch hydrolysis is indicated by a clear zone due to the action of amylase.
Tributyrin Agar is used to show fat hydrolysis by the enzyme lipase. Organisms that can hydrolysize fat will have a clear zone around their colony.

24 Test Tubes Inoculating.....

Today we inoculated tubes of sucrose, lactose, glucose and nutrient agar with E. coli, Bacillus subtilis, Staph. aureus and our unknown. We also did a quadrant streak on Tributyrin Agar and Starch Agar with E. coli, Bacillus subtilis and our unknown. I still think my known is Staph aureus, but have done little in the last month or so to progress beyond my intial guess.

Mucho Media!

This test indicates that I test negative for dental caries at both 24 and 48 hours. This explains why I never have cavaties. Lucky me, I've inheritted my father's good teeth. My brother on the other hand would probably have a yellow tube at 24 & 48 hours. This means he has marked risk for dental caries. He has my mother's bad teeth or should I saw destructive mouth flora? He has frequent cavaties and problems with his teeth. Count you blessings if you have a green tube!
Here is a face skin culture on a Blood Agar plate. Microbe shows Gamma-Hemolysis (none) created small white circular colonies. Organism is most likely non-aureus Staph, which is considered normal skin flora. Skin from my arm on blood agar. 2 microbes arose. 1 was Beta-hemolytic (complete lysis) and is the large yellow growth. The other was Gamma-hemolytic (no lysis) and is the small round white colonies. The first microbe is most like bacillis normally found in the soil but can sometimes be found as skin flora. The second is most likely non-aureus Staph. Both are considered to be normal skin flora.

Today's Lab Interpritation

Tuesday in lab we inoculate 3 tubes of Thioglycollate broth to determine the oxygen requirements of our unknowns. We inocullate the tubes with E. coli, Clostridium p. and our unknown. We also did a similare exercise with nutrient broth plates using E.C. and C.P. as our controls to compare our unknowns to.

Results:
E. coli: facultative
C.p: strict anaerobic
#30: facultative

Plate Interpritation:
E. coli: facultative
C.p: anaerobe
#30: facultative, with smooth edges and made the medium a slightly darker color.

From the inoculated broth or our working slants we where to make slides for staining and viewing to determine the shape of our unkown as well as the E. coli and Clostridium p.

My slides made from the slant were a little hard to examine. I remade 3 more from the broth but didn't have time to look at them. I did accidently stain all 3 rather than just one. Hopefully I can use these next week.

Microbe Shapes:
E. coli: bacillus
C.p: cocci clusters
#30: Dr. Belcher thought perhaps mine where cocci that formed chains. I couldn't tell becuase my smear was a little thick.

Next week it's gram staining action like you've never seen!

This Weeks Media

Eosin-Methylene Blue is used to select G- lactose fermenters. The selective agent is eosin and methylene blue. The differential agent is methylene blue and lactose. If the organism ferments lactose it is most likely G- and lactose +. Medium is used to grow enteric microbes.
E coli: Microbe tolerates EMB. Microbe ferments lactose. Microbe is most likely G-
S. Aureus: Microbe does not tolerate EMB. Microbe does not ferment lactose. Microbe most likely G+
Ps. aeroginosa:Microbe tolerates EMB. Microbe does not ferment lactose. Microbe most likely G-
Unknown #30: Microbe does not tolerate Eosin & MB. Does not ferment lactose. Most likely G+

Phenyl Ethyl Alcohol is a medium used to select G+ cocci. The selective agent is phenyl ethyl alcohol and there is not a differential agent. Organisms that show good growth on this medium are most likely G+.

E. coli: Microbe does not tolerate Phenyl Ethyl Alcohol. Most likely G-

Ps. aeruginosa: Microbe does not tolerate PEA. Most like G-

S. Aureus: Microbe tolerates PEA. Most likely G+

Unknown #30: Microbe tolerates PEA. Most likely G+

MacConkey Agar is a medium used to select G- lactose fermenters. The selective agent is bile salts and the differential agent is neutral red and lactose. Lactose + microbes appear pink-red. Lactose - microbes appear cream to colorless. This medium is used to grow G- enteric microbes. E. coli: Microbe tolerates bile salts & Crystal Violet dye. Does ferment lactose. Most likely G-

Ps. Aerug: Microbe tolerates bile salts & CV. Does not ferment lactose. Most likely G-

S. Aureus: Microbe does not tolerate Bile salts or CV. Does not ferment lactose. Most likely G+
Unknown #30: Microbe does not tolerate bile salts or CV. Does not ferment lactose. Most likely G+

Mannitol Salt Agar is a medium used to select mannitol fermenting staph microbes. The selective agent is sodium chloride and the differential agent is phenol red. Positive mannitol fermenters turn yellow.
E coli: Microbe does not tolerate Sodium Chloride. Does not ferment Mannitol. Most likely G-

Ps. Aerug: Microbe does not tolerate NaCl. Does not ferment Mannitol. Microbe most likely G-

S. aureus: Microbe tolerates NaCl. Microbe ferments Mannitol. Microbe most likely G+

Unknown #30: Microbe tolerates NaCl. Microbe ferments Mannitol. Microbe most likely G+

Summary:

Unknown #30 had similare growth and fermentation patterns as S. aureus. However, unkown #30 had slightly better growth and fermentation when compared to S. aureus.

Unknown #3o tolerates MSA & PEA and does not tolerate EMB & MAC. It tolerates Phenyl Ethyl Alcohol, NaCl and ferments Mannitol. This microbe is most likely Gram + and tolerates a salty environment.

Inerpreting Growth on Media

1. Purpose/ characteristics of media?
a. selective, differential, both
b. what are the agents
c. types of microbes typically isolated

2. Is there good growth?
Yes: "Microbe tolerates_(selective agent)_" No: "Microbe does not tolerate_(selective agent)_"

3. Is there a change in color?
Yes: "Microbe ferments/uses/poseses_(differential agent)_" No:"Microbe does not ferment/use/poses_(differential agent)_"

4. Microne is most likely "__G-/G+__"

Lab #6

Previously in lab we made a 4 section streak plate on 4 different types of growth medium.
We divided each plate into 4 section like a pie and in each section drew a line from 4 different microbe broths.
Media:
Eosin Methylene Blue Agar
Phenylethyl Alcohol Agar
MacConkey Agar
Mannitol Salt Agar

Microbes:
Unknown #30
E. coli
Staphyloccocus aureus
Psudomonas aeruginosa

E. Coli

Microbe:
Escherichia coli

Common Name:
E. coli

Oxygen requirements:
Facultative (Anaerobic or Aerobic)

Origin:
Intestines of mammals

Day 3 #30

This is my first Quadrant Streak plate and I'm rather proud of it. I had to redo it 3 times because I kept streaking from 1 instead of 3. My unknown #30 made neat little colonies in the middle.

Here are it's stats:

Growth Medium:
Nutritive Agar
Margin:
circular or punctiform
Consistency:
moist maybe slime
Pigment:
none
Odor:
smelled like dog poop (no joke)

Day 2 with #30

This is my working slant of the unknown #30. Next to it is my unknown in a differential broth. You can't tell from this picture, but my unknown is facultative. This means it utilizes both aerobic and anaerobic respiration.

Day 1 with Unknown #30

We made working slants of our unknown. That was pretty musch it. Hopefully I did it right and next week I'll see my microbe growing on the agar slant.

Flame the Loop

It is rather evident that this is my first Microbilogy class. Infact, it's my first Biology class since Highschool, which was over 10 years ago. In highschool Biology we touched apon amebas and looked at yeast eating sugar. We never did petri dishes and innoculations. Needless to say this is all Greek to me. Most of the genis names are Greek come to think of it. For example Staphalococcus means bunch of grapes in Greek.
The picture on the left was my artistict interpritation of the Sterile Innoculation process as outlined in my lab manual.
For those of you already in Micro or Lab Techs looking for a laugh, you will see that my ideas are a bit laughable.
I was thinking old school as to what the "loop" should look like.