Outline. This week s plans Introduction to Kirby Bauer and Antibiotics Introduction to Biochemical analysis of strains

Transcription

1 Outline This week s plans Introduction to Kirby Bauer and Antibiotics Introduction to Biochemical analysis of strains 1

2 This week 2

3 Monday Finish Exercise 10, part C. Run MspI restriction digest on a gel and see how it looks Perform PCR clean up for sample to sequence We want 1 DNA sample to sequence/pair Pull out the strain that you are sequencing from the glycerol frozen stock onto Nutrient Agar We will do Kirby Bauer (Ex. 9) and Biochemical Characterization on that You need to come in on Tuesday and start overnight cultures in LB Broth 3

4 Tuesday Go to the lab before 1 pm or 4-5 pm to start an overnight culture of your food strain (one/pair). Lab manual says there are several plates of bacteria, but that is not the case. You should use your food lab bacterium. Remember how to start a culture? One single colony >> LB tube Sterile Shaking 37C 4

5 Weds-Kirby Bauer (Ex. 9) and Biochemical characterization (11) Both of these experiments will tell you more about your food strain. 5

6 Kirby Bauer Swab your culture onto Mueller Hinton Agar Swab E. coli control onto Mueller Hinton Agar Add antibiotics to each sterile disk and place disk on plate Template at end of exercise to help with placing discs. Need all 8 on each plate. Incubate until next day at 37ºC (staff will grab all plates) X / jpg 6

7 Kirby Bauer: Results Measure and record diameter of clearing zone Note whether zones are totally or partially clear, or even contain small colonies that may be resistant mutants 7

8 Antibiotics target crucial bacterial processes We are using: Ampicillin, Chloramphenicol, Ciprofloxacin, Kanamycin, Nalidixic Acid, Rifampin, Streptomycin and Tetracycline 8

9 Physiological Analysis of Unknowns (Ex 11): Weds You will be working with the same strain as the one you isolated DNA from. The goal is to learn a bit more about the strain. With your plates from Monday, note the colony appearance. Perform a gram stain Set up the following tests. 9

10 Set up these tests Motility Test Put wooden stick into culture, and then stab into soft agar. Carbohydrate utilization Inoculate the following: Phenol red + glucose broth w/durham tube Phenol red + lactose broth w/durham tube Phenol red + mannitol broth w/durham tube (Use stick dipped into culture) Indole Test Inoculate as above Tryptone Broth. This tests for the production of indole from tryptophan. Tryptone is a peptone that contains high concentrations of tryptophan. 10

11 Next Monday you read and complete the previously set up tests Test Colony morphology Procedure and Interpretation Using aseptic techniques, examine with a dissecting scope, and record observations. Motility test Carbohydrate utilization Indole test Catalase test Oxidase test Look for indications of growth spreading beyond the point of inoculation. Yellow = positive Red = negative Bubble in durham tube = positive for gas production Add 10 or 12 drops of Kovac's reagent. Pink layer on top = positive Place 2 to 3 drops of 3.0% H 2 O 2 on colony growth (expendable) Formation of bubbles = positive Place 3 to 4 drops of oxidase reagent (1.0% dimethyl-pphenylenediamine hydrochloride) on a piece of Whatman filter paper in a Petri dish, and smear cells from colony growth on the paper Color change to dark red/black = positive 11

12 The carbohydrate utilization tests are based on acid production during fermentation Different sugars can be used to fuel fermentations End products are acids and gases Some microbes just can t use certain sugars--they lack the transporters or ability to break them down 12

13 Indole Test tests for Tryptophan degradation Some microbes have the ability to degrade tryptophan to indole Indole is detected by adding Kovac s reagent (isoamyl alcohol, p- Dimethylaminobenzaldehyde, concentrated hydrochloric acid) Bacteria that test positive for cleaving indole from tryptophan include: Aeromonas hydrophilia, Bacillus alvei, most Citrobacter sp., Edwardsiella sp., Escherichia coli, most Proteus sp. (not P. mirabilis), and Vibrio sp. Indole-Negative Bacteria: Actinobacillus spp., Aeromonas salmonicida, most Bacillus sp., Lactobacillus spp., most Proteus mirabilis, Pseudomonas sp., Salmonella sp., Serratia sp., Yersinia sp. 13

14 Catalase Test tests degradation of hydrogen peroxide Some microbes produce an enzyme that can cleave hydrogen peroxide If bubbles or froth forms, the organism is said to be catalasepositive. Staphylococci and Micrococci are catalasepositive. If not, the organism is catalasenegative. Streptococci and Enterococci are catalasenegative. 2 H 2 O 2 2 H 2 O + O 2 14

15 Oxidase Test:Tests for presence of certain cytochrome C oxidases Place 3 to 4 drops of oxidase reagent (1.0% dimethyl-pphenylenediamine hydrochloride) on a piece of Whatman filter paper in a Petri dish, and smear cells from colony growth on the paper. Color change=positive Oxidase Test: A. Pseudomonas aeruginosa - Purple color indicates positive. B. Escherichia coli - Negative. 15

16 The rest of the quarter Monday 11/14: No morning lecture (we ve covered it all) but of course there is lab class in the afternoon. Ottemann will have office hours during that time. Weds 11/16: Group projects only, no other experiments. Attendance is optional. Monday 11/21: Lecture on bioinformatics and discussion of lab practical format. Monday 11/21: Bioinformatic analysis in groups of 8 in the Thimann labs computer room. Please bring your lab top if you have one! Exercise 9 (Kirby Bauer) due Weds 11/23: Group projects only, no other experiments. Attendance is optional. Monday 11/29: No lecture, office hours Monday 11/29: Group projects only, no other experiments. Attendance is optional. Weds 11/30: Lab practical. Tuesday 12/6: Group project presentation. Powerpoint. 15 minutes/group, which is about 15 slides