Unknown day 3

7/27/12

*Made 2 slides, one from each slant to assure isolation
- All pink on one slide and all purple on the other.
*made 2 more slants and incubated

Unknown day 2

7/26/12
*checked my streak plates for any definite difference between any colonies.
- the Mac agar had pink growth is +pos for lactose fermentation
the Mac also inhibits growth of gram + so i isolated a neg from that
-The blood agar inhibits gram -neg so i pulled my gram + pos from the blood agar
*I made 2 more gram stains to see if i had isolated them, but didnt get a good slide to come out!
-So i went ahead and started 2 sepreate broths for gram+ & gram-

Unknown day 1

7/25/12
*Gram Stain to see what morphology my bacteria are
-i seen 2 groups of bacilli- short rods and longer rods which were forming chainlike groups
*i streaked my plates for isolation
-1 MacConkey agar
-1 Sheep blood agar
-2 TSA

FIRST GRAM STAIN/UNKNOWN

MacConkey Agar

MACCONKEY AGAR TEST:
*Medium contains lactose,bile salts, neutral red, and crystal violet.
-to grow and check gram- neg bacteria for lactose fermentation
inhibitor: bile salts & crystal violet inhibits gram +pos growth
pH indicator: neutral red dye
* lactose fermenters will turn the agar red or pink for a pos+ result
-we streaked 2 agar plates sarting in section 1-4 incubated for 24 hours
*E. coli had good growth-red is a lactose fermenter
*S. epi had poor growth no ferm.
*S. enter had good beige growth no lactose ferm
*E. aero good red growth- ferments lactose

Mannitol Salt

MANNITOL & SALT:
*method used for isolating gram +pos cocci
and for identifying S. aures from other staph
*Indicator- phenol red
*Inhibitor- NaCl
-we divided the agar in half and inoculated each side with a diff germ
-incubated for 24 hrs
*S. aures- turned yellow with a halo around it +pos for mannitol fermentation
*S. epi- turned pink without halo is a -neg for ferm mannitol

Blood Agar

BLOOD AGAR:
Medium-sheep blood & triptic soy base
*test for hemolytic of RBC's ability of gram +pos cocci
we took 2 plates and streaked them with organisms
incubated for 24 hours
*S. aure had raised dots-B hemolytic
*E. faec had lightly raised dots- Y hemo
*S. pneu had dots- A hemo

Eoisin Methylene Blue

Eoisin Methylene Blue:
*promotes growth of gram -neg organisms while inhibiting gram +pos
inhibitor: eoisin, methylene blue
we streaked every corner with a different org.
*E. coli-growth lactose fermenter,
*S. epi- no growth cuz gram -
*S. ente- growth lactose ferm
*E. aero- pink growth lactose ferm

PhenylEthyl Alcohol agar

PHENYLETHYL ALCOHOL:
*selective for gram +pos.
-inhibitor- phenylethyl alcohol inhibits gram -neg
interferes with DNA synthesis
divided the agar into 3 sections and streaked each section with a diff organism
E. coli- no growth, E. faec-growth clearing B hemolysis, S. aur- growth some white

Columbia CNA

COLUMBIA CNA :
*agar to promote gram +pos organism growth
-Inhibitor: antibiotics colistin nalixic acid CNA inhibits gram -neg growth
good growth is clearing-B hemolytic
greening good growth- a hemolytic
no growth same color - Y hemolytic

Methyl Red & Voges Proskauer Test

METHYL RED: (M.R.)
*M.R. part of test is used to detect organisms capable of mixed acid fermentation.
-medium contains peptone & glucose with a phosphate buffer and pH indicator
Indicator: Methyl red dye
* the solution turned red with a +pos result and yellow is -neg.
VOGES- PROSKAUER: (V.P.)
*for organisms that can ferment glucose and convert acid products to acetoin & butanediol
we added vp reagents after incubation it turned red for pos+ result
we used E. coli & E. aero.
following incubation we transferred part broth into two other tubes for VP test
E. coli was +pos for MR & E. aero was +pos for VP.

Phenol Red Broth Test

PHENOL RED BROTH:
*used to detect organism's ability to ferment in after a carbohydrate is added to test.
-indicator: Phenol red dye
-pH rises color turns pink is deamination of perptone and a neg.- result for carb hydrolysis.
-pH lowers it turns yellow a pos+ result for mixed acid fermentation
*3 tubes were used with phenol broth & durham tube for detection of gas production.
-inoculated with 3 organisms./ 24 hrs incubate
- A. faec,-no fermentation remained red
 E. faec-pink broth for peptone usage
 E. coli-yellow broth with a bubble in tube +pos for ferm

Tryptone test

TRYPTONE TEST:
*checking for the enzyme cysteine desulfurase a sulfur reduction enzyme. also thiosulfate reductases
for bacteria producing indole is SIM medium is used for. this test is a series of tests along with Indole, Methyl Red. Voges-Proskauer, and Citrate
*we stab inoculated 2 broths with a needle and incubated for 24 hours
we used E. coli & E. aero
*examined the stabline for spreading and black precipitation in the medium
the E. coli changed color and broke down indole to pyruvate is +pos for enzyme
the E. aero is -neg for enzyme no color change

Citrate test

CITRATE TEST:
*checking for the enzyme citrate permease that convert molecules in a cell to pyruvate
*3 citrate slants one with E.coli, E. aero, and 1 control uninoculated
with the needle we streaked the tops of the slants with zig-zag pattern
incubated for 24 hours
check for blue color change E. coli was green no color change -neg for citrate permease
E. aero changed blue +pos for enzyme activity

Oxidation-Fermentation test

OXIDATION FERMENTATION TEST:
*looking for aerobically & anaerobically or fermentaion of bacteria's ablility to utilize glucose
* we used hour glass shaped tubes with mineral oil seperating the middle so that oxygen cannot get to bottom. the medium is enriched with (O-F) high sugar-to-peptone ratio
its prepared with glucose, lactose, sucrose, maltose, mannitol, or xylose
*inoculated 3 with E. coli, P. aeru, A. faec with a needle and stabbed all the way 1/2 cm from the bottom of the butt. Incubated for 24 hrs.
the part that turns yellow is the oxidizor if turned blue its non-reactive it deaminated the peptone
if yellowe on top and bottom its a fermenter
E. coli-fermenter,  P. aeru is yellow only on top oxidizer, A. faec wuz non-reactive

Triple Sugar Iron test T.S.I.

TSI TEST:
*checking aerobically & anaerobically for glucose, lactose & sulfer fermentation.
6 slants with E. coli, P. aureus, salmanilla, A. aero, E. faec, E. aero
we zig-zag pattern each one after stabbing down 1/2 cm from butt of tube
*the black slant is + pos for sulfer
*the yellow/yellow e.coli is acid fermentation for glucose & lactose
*the yellow one that cracked and lifted up is pos+ for gas production
*if no change in butt and red slant it is aerobically alkalinity

Coagulase test

COAGULASE TEST:
*checking for the presence of enzyme coagulase which is a clumping factor
used 2 plasma of tubes & inoculated  with S. aure & S. epi
* incubated for 24 hours
the S. epi had no presence for clotting is - neg. for coagulase
the S. aure was +pos for clotting enzyme

Catalase test

CATALASE TEST:
*to check for enzyme activity of catalase in organism
took a slide & placed a good size of bacteria in two diff spots-
one S. epi & the other E. faecalis
* then place aseptically (not the metal loop) 1-2 drops of H2O2 hydrogen peroxide
on both specimens and watch for bubbles
* the S. epi bubbled quickly & E. faecalis did not. bubbles are a +Pos reaction for catalase

Urea hydrolysis test

 UREA HYDROLYSIS TEST:

* test for enzyme urease
3 urea broths one uninoculated one for control, E.coli, P. vulg in the other 2 broths
incubate for 24 hours . coli
examined for color change. phenol red is a pH indicator used

the P. vulg was pink pH above 8.5 it turned pink +pos for urease
E. coli pH was low turned yellow -neg for enzyme

Starch hydrolysis

STARCH HYDROLYSIS:
*to test for presence of enzyme amylase
* used a starch agar plate and divided it in two with pencil mark
put B. sub in one side and E.coli in the other
incubated for 24 hours
added drops of iodine to dish
the side that rejected the iodine has a clear path around the germ B.sub is +pos for enzyme
E.coli was -neg for amylases starch digesting enzyme

Gelatin hydrolysis

GELATIN HYDROLYSIS TEST:
*test for the presense of gelatinase enzyme
*2 broths added P.aerug & E. coli
incubated for 24 hours
chilled on ice for 30 min. and removed from ice- let room temp affect the tubes,
P. aerg liquified +pos for gelatinase
E. coli stayed gel-like -neg for enzyme

Nitrate reduction test

NITRATE REDUCTION:
*to test for enzyme nitrate reductase
3 broths A. faecalis, E.coli, P. aerug
incubated for 24 hours
*look for gas bubble in all 3 broths- was present in A. faec +pos for NO - N2O - N2
*added reagent A & B to remaining broths- color change to red E. coli +pos for enzyme
next added zinc to remaining broth turned red P. aerug is -neg for enzyme

Oxidase Test

OXIDASE TEST:
*to test for bacteria containing enzyme cytochrome C oxidase
we put E. coli on one cotton swab & A. faecalis on another swab
added 2 drops of reagent to each swab
color change within 30 seconds-
*E. coli had no change -neg for enzyme
*A. faecalis changed to blue-purple is +pos. for enzyme

Streak & Spread plates

7/16 STREAK & SPREAD PLATES
2 spread and 3 streak
*for streak we sectioned the back of the agar by china pen in 4 divisions numbererd 1-4.
-we started in area 1 placing loop of organism in a zig-zag pattern.
then burned loop and drug 1 line of org. to #2 section then did a zig-zag motion again.
so forth so on to all 4 sections.
*for the spread plates we placed dime sized drop of broth onto the agar
and used a u-shaped tool to spread evenly around germ.
*we flipped the streak plates upside down to prevent condensation in incubation-24 hrs.
7 different organisms were used.
*S. epi  *S. marcescens  *P. aeru. *P. mirabilis  *M. luteus  *E. Faecal  *B. sub

Acid-fast & Endospore staining

July 10th, Acid-Fast and Endospore Staining-
Acid-Fast, 3 slides, S. epi. as control and M. smeg. for the acid-fast cell.
Over a steamer- carbolfuchsin for prim. acid-alcohol for decolorizor, brilliant green for counterstain.
S. epi. blue/grn cocci and M. smeg. reddish bacilli-rods.

Endospore- 3 slides, S. epi. as control and B. sub. as the spores.
Over a steamer- Malachite green for prim. & rinsed w/water & counterstained with safranin.
Spores stained green- big eliptical shapes. & vegetative n daughter cells stained pink-red.

Gram Stain

July 6th, Gram Stains-
stained 2 slides, S. epi. -neg chrg. E. coli. +pos chrg. bacteria.
S. epi. wuz Gram pos.+ purple clusters of cocci, E. coli. wuz Gram neg.- pink bacilli-rods.