Worksheet for Unknowns Identification

You were given a test tube with a mixed culture of 1 Gram-positive cocci and 1 Gram negative bacilli. Your task is to isolate these organisms and identify these microscopically as well as by using biochemical tests. Assume that you performed this experiment in lab for 4 days.

Below is the worksheet of what you did on each day. The worksheet has questions at every step which should be answered. You will also be asked to pull appropriate pictures from the pictures bank and copy and paste at various points. You will have to fill out tables, write your observations/results in the tables and write out interpretations/conclusions for each test. Finally, compare your results to a Gram positive and Gram-negative identification chart that will be provided to you and make a conclusion about the name of the Gram-positive and Gram-negative microbe that was in your mixed culture tube.

When you are done, please e-mail this file back to me with the answer and pics in place. If you feel that this Word doc is too cumbersome, please feel free to make a PPT that has the same information. It’s a little easier to move pics around with PPT. This will be due by Next Tuesday, April 21, 2020.

Day 1 - You were given a mixed culture tube and performed a 4-way streak on MacConkey and CCNA agar. You incubated the plates at 30° for 48 hours. The CCNA plate was put in Gas Pak jar before incubating.

1. What was the reason for doing a 4-way streak on the two agars?

2. What would be the objective for using MacConkey agar and CCNA agar in particular?

3. Why was CCNA put into Gas Pak jar?

Day 2 – You performed the following 3 steps:

Step 1. Observe your results for MacConkey and CCNA agar from Day 1 (Cut-paste your pictures here).

MacConkey agar Pic CCNA agar pic

Write your observations of each plate in the table below

Table 1 – Results for streaking on plates

Color of colonies Isolated colonies (y or n)MacConkeyCCNA

Step 2. Results were obtained after doing a Gram stain from MacConkey and CCNA.

a. Cut-paste pictures here and fill out the results table for Gram stain below.

Place pic of CCNA Gram stain here Place pic of

MacConkey Gram stain here

.

Table 2: Gram stain results

Cell shape observations

Gram stain results

MacConkeyCCNA

b. Answer questions on Gram stainingB - 1. What is the role of crystal violet?

b- 2 – Name the mordant in this process.

b- 3 – What is the decolorizer and what does it do to Gram-negative cells?

b-4- What would happen if you forgot to add safranin while performing Gram stain from the MacConkey plate?

Step 3. Culture from MacConkey agar was inoculated onto Gram-negative working stock slant. Culture from CCNA was inoculated onto Gram-positive working stock slant. Both tubes were incubated at 30°C for 48 hours. Cut-Paste pictures of working slants/stock below.

Day 3. – Cultures from the working stocks were inoculated into various biochemical media for Gram-positive and Gram-negative identification.

Gram positive identification

1. Catalase test was done on culture from Gram-positive working slant. Place picture of catalase test below and answer questions.a. Insert pic of the catalase test result below.

b. Observe and interpret whether it is Staphylococcus or Streptococcus. Give reason for such an interpretation.

c. What reagent had to be used to perform the catalase test?

Based on your result in 1-b you took media for either Staphylococcus or Streptococcus/Enterococcus. You should pick either question 2 or 3 to answer and proceed.

2. For Staphylococcus: You inoculated a tri-plate and incubated it for 48 hours at 30°Ca. What 3 agars are present in the tri-plate?

b. Which agar do you use for antibiotic sensitivity testing and what antibiotic was used?

3. For Streptococcus: you inoculated a blood agar plate, a mini MSA plate and a BEA slant. All 3 media are incubated for 48 hours at 30°C.a. Which 2 agars would you put in Gas Pak jar before incubating?

b. On which agar will you perform antibiotic sensitivity testing and which antibiotic/s were used?

Gram-negative identification

1. You inoculated 7 different pieces of media for testing Gram-negatives: TSI, urea, citrate, SIM, LIA, MR/VP and nitrate. You will incubate all of these at 30°C for 48 hours taking care that caps are loose.

a. What 3 sugars are present in TSI and what are their percentages?

b. What enzymes do you want to test in the urea tube, nitrate tube and citrate tube inoculations?Urea –Nitrate-Citrate -

c. What 3 things would you want to test in SIM agar?

d. What acid/s are we testing for in MR test?

e. In Lysine Iron agar what 3 processes are being tested?

Day 4. On the final day results for Gram-positive and Gram-negative results were observed and interpreted. The unknown Gram-positive and Gram-negative results were compared to a chart given (attached) and the closest match was obtained.

Gram-positive

Observe your Gram-positive inoculations (cut-paste all pictures for Gram-positive inoculations below from the picture bank provided).

Table 3: Gram-positive results and interpretations

Staphylococcus

Result

Interpretation/conclusion Strep/Entero

Result Interpretation/conclusion

MSA fermentation

Hemolysis

DNASE Bacitracin

Novobiocin SXT

BEA

MSA growth

Compare your results to the Gram-positive identification chart provided and conclude your Gram-positive organism.

Gram-negative

Observe your Gram-negative inoculations (cut-paste all pictures for Gram-negative inoculations below from the picture bank provided).

Spring 2020

Triple Sugar Iron agar Urease

Citrate

SIM Deep MR VP Nit LIA

Gram-negative bacterium

Slant Butt Gas H2S Indole H2S* Motility

Decarb

Deam

Table 4. Gram-negative results/observations

Write your interpretations/conclusion for each of the tests above.

TSI –

Urease-

SIM deep –

MR and VP-

Nitrate –

LIA –

MacConkey Agar -

Compare your results to the Gram-negative identification chart provided and conclude your Gram-negative organism.

Final conclusion:

Gram-positive: __________________________

Gram-negative: _________________________

Reference charts – Gram-positive and Gram-negative Identification

Staphylococcus Identification reference chart

Organism MSA Fermentation DNase Novobiocin diameter (S/R)

Staphylococcus intermedius

POS POS S-30

Staphylococcus saprophyticus

POS NEG R -12

Staphylococcus sciuri POS POS R -15

Staphylococcus capitus NEG NEG S - 25

Streptococcus/Enterococcus Identification reference chart

Organism Hemolysis Bile Esculin

MSA Growt

h

SXT Diameter/S

or R

Bacitracin Diameter/S

or RStreptococcus equi Beta NEG NEG R S - 18Enterococcus faecalis

Gamma or alpha POS POS R R

Streptococcus uberis

Gamma or alpha POS NEG R S-17

Streptococcus gallolyticus Alpha POS NEG R R

Triple Sugar Iron Agar

Ure

ase

Citr

ate

SIM Deep

Nitr

ate

Lysine Iron Agar

MaC agar

Gram-negative Bacterium

Slan

t

Butt

Gas

H 2S

Indo

le

H 2S*

Moti

lity

Slan

t Lys

ine

Deam

inati

on

Butt

Lys

ine

Lact

ose

Ferm

enta

tion

Edwardsiella

tardaAlk A - + - - + + + + - + - +

-

Enterobacter

aerogenesA A + - - + - - - - + + - +

+

Enterobacter

cloacaeAlk A + - - + - - - - + + - +

-

Escherichia

coli A A - - - - + - + + - + - +

+

Klebsiella

oxytoca A A + - - + + - + - + + - +

+

Klebsiella

pneumoniae A A - - - - - - - + + + - +

-

Morganella

morganii Alk A - - + - + - + + - + - -

-

Proteus

mirabilisAlk A - + + + - + + + - + + -

-

Gram-negative identification reference chart

Picture Bank-You will use these following pictures to complete the unknown worksheet below

35mm

LIA

Catalase test