Transcript Lab18

Lab 18 Goals and Objectives:
Exercise 21: Enumeration
Work as two pairs per group (max 5 groups)
Both pairs use same Escherichia coli culture
Use pre-poured plates: no molten agar pour plates
Divide work: One pair does Standard Plate Count, other does
Absorbance Readings
Plate count pair needs:
Absorbance pair needs:
3 – 99ml bottles sterile water
4 nutrient broth tubes
4 pre-poured plates
5 cuvettes
4 tubes of beads
10ml pipettes
(use beads to spread bacteria on plate)
1ml pipettes
For absorbance readings, make the required dilutions (1:2, 1:4 1:8, and
1:16) in the nutrient broth tubes and then transfer ~4ml of each to a
separate cuvette for reading in the machine. Cuvette #1 is for the
original undiluted culture (1:1).
Fig 21.1
*Label the plates with the dilution factor!
Fig 21.9
1. Make dilutions in culture tubes
2. Put 4ml of
original culture in
a cuvette, label it
1:1
3. Transfer ~4ml
each dilution to a
cuvette labeled
with dilution (1:2,
1:4, 1:8, 1:16)
4. Read absorbance number for all 5 in spec beginning with most dilute
To save time:
1. 4ml culture into 1:2 culture tube with broth, mix with pipette
2. Suck up whole volume (8ml) with new pipette, dispense 4 ml into 1:4
broth culture tube and remaining 4 ml into 1:2 cuvette.
3. New pipette, mix 1:4 culture tube, suck up whole volume, dispense
4ml into 1:8 broth culture tube and remaining into 1:4 cuvette.
etc.
Lab 17 Goals and Objectives:
Exercise 21: Enumeration
Work as two pairs per group (max 5 groups)
Both pairs use same Escherichia coli culture
Use pre-poured plates: no molten agar pour plates
Divide work: One pair does Standard Plate Count, other does
Absorbance Readings
Plate count pair needs:
Absorbance pair needs:
3 – 99ml bottles sterile water
4 nutrient broth tubes
4 pre-poured plates
5 cuvettes
4 tubes of beads
10ml pipettes
(use beads to spread bacteria on plate)
1ml pipettes
For absorbance readings, make the required dilutions (1:2, 1:4 1:8, and
1:16) in the nutrient broth tubes and then transfer ~4ml of each to a
separate cuvette for reading in the machine. Cuvette #1 is for the
original undiluted culture (1:1).