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).

*Label the plates with the dilution factor! Fig 21.1

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 Fig 21.9

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).