Viable Population Count Micro-Lab Subject of Study 5

Pour Plate procedure

I. Materials Involved: Media:

1. Tube of broth (E.coli)  E.coli broth
2. 7 “labeled” tubes of sterile saline  Melted Agar
3. 8 sterile pipettes 1ml  Tubes saline
4. 1 blue pipette pump Instruments:
5. 6 “labeled” sterile, empty, petri dishes  Pipettes
6. 12 tubes of melted agar (must be held @ 45°C)  Blue pipette pump
 Petri dishes
II. VP Count
1. Count the colonies on each plate
2. Choose counts between 30-300
3. Adjust sample volume for a ml sample, if necessary Perform aseptic transfer.
4. Determine concentration of original broth sample Avoid touching sides of
tube and/or saline w/
pipette
1.0ml

6
2 3 4 5 7
1
1st
E. Mix
coli
10-5
10-1 10-2 10-3 Pipette 10-4 10-6
Pipette
6
7 Pipette
1.0
ml 1.0
ml Pipette 8
Pipette 1.0
A 7 ml
6 0.1
Pipette 5A 6A
0.1 ml
5
ml
0.1 ml 5B 6A

Colonies are counted by:

• Direct observation
• Quebec colony counter
• Considered (TNTC)

Total Cell Count: live and dead cells vs. Viable Cell Count: only live cells

Formula:
Sample Volume: # Colonies x D.F. (delusion factor) = # Cells/ ml
Ml
 Micro-Lab Subject of Study

Tube Sample Vol. DV Count DF

Labeled
4 0.1 ml 10 -4 TNTC 10 4
5A 1.0 ml 10 -5 920 10 5
5B 0.1 ml 10 -5 130 10 5
6A 1.0 ml 10 -6 110 10 6
6B 0.1 ml 10 -6 18 10 6
7 1.0 ml 10 -7 8 10 7

130 colony x 10 1300 colony x 105 1300 x 105 cell/ml 1.3 x 10 8 cell/ml
Example 0.1ml x 10 1.0 ml
Count: 6B

carted result multiplied by 10 count decimal solution

Example 18 colony x 10 180 colony x 106 180 x 106 cell/ml 1.8 x 108 cell/ml
Count: 6B 0.1 ml x 10 1.0 ml

Only use the colony count that initially falls between 30 and 300
Diluted to much 30 and 300 Not diluted enough
It is only assumed each bacteria grows and divides.
 Atmospheric Oxygen RequirementsMicro-Lab Subject of Study 6

Medium
• Broth Thiogloycollate Clostridium sporogenes
The amino acids and glucose in the medium can be respired, and glucose is a fermentable
source for organisms as well as Clostridium which can ferment amino acids.

Equipment Used:
Bunsen burner
Inoculating loop
Test tube rack
Pasteur pipettes

The observed growth patterns of organisms in this medium determine their oxygen relationship
designation, which brings the abilities of respiration, fermentation and the catalase reaction and
also whether there is an inhibitory effect on the organism in the presence of air.

Top

Middle More
Area Even

Less Bottom

Obligate Microaerophillic Facultative Aerotolerant Obligate Aerobes

Aerobes Anaerobes Anaerobes
Requires O2for growth Greater growth in the Only anaerobic
growth. Occurs where presents of O2, but growth.Ceases in
high concentrate of O2 can occur throughout presents of O2
has diffused into medium tube.

Needs limited amount of O2 Growth occurs evenly

for Growth. Occurs where O2 has no effect.
high concentrate of O2 Anaerobic growth that
low diffused into medium continues in O2 presents

Peroxide anion: Anaerobic Culture Method

2H2O2 catalase 2H2O + O2 Reducing Media
• Contain chemicals thioglycolate that
combine O2
H2O2+2H+ peroxidase 2 H2O
• Heated to drive off O2
• Provides Goldish Color
 Micro-Lab Subject of Study

Anaerobic Jar
This system is a temporary method for
the exclusion of O2 from a sealed jar
used for incubation of anaerobic
cultures in a non-reducing medium.
CO2 H2
• Duplicate sets of cultures are made
Anaerobic indicator
(methylene blue)
• A single-line steak inoculation, of
Hydrogen Petri plate each test organism
Gas Pack (Inoculated)
• With corner of generator, insert
generator
inside gas pack jar (one set of
cultures in a inverted position)
When water is mixed with chemical pack, hydrogen
and carbon are generated • 10ml of H2O is added (with pipette)
Hydrogen and atmospheric oxygen in the jar combine to gas generator, quickly seal cap
to form water Removing Oxygen
Indicator shows no color when O2 is removed • Jar is then incubated @ 37°F for 24-
48 hours