BIO 260: Lab Quiz #2

How does the pGLO plasmid work??? [TEXT BROKEN DOWN INTO STEPS]

1. ARABINOSE. The simple sugar arabinose is present in the bacteria's environment. 2. AraC. A promoter region upstream of these genes acts as a molecular on/off switch that regulates their expression. 3. SWITCH ... When the Arabinose Promoter is turned on, instead of arabinose production starting, the GFP Green Fluorescent Protein) gene switches on. 4. When bacteria transformed with pGLO plasmid are grown in the presence of arabinose, the GFP gene switches on, causing the bacteria to express GFP and fluoresce brilliant green.

Serial Dilution and Pour Plate Method - results - 1

1. Count the number of colonies on each plate. For each plate calculate the number of organisms per milliliter of milk. Average the four figures and report a final plate count. If the number of colonies on a plate is too numerous to count (more than 300 per plate), select those plates that have a manageable number to use for your calculations.

Serial Dilution and Pour Plate Method - 1

1. Get four tubes of melted nutrient agar from the hot water bath.

E. coli transformation and antibiotic resistance - 1

1. Label one closed micro test tube +pGLO and another -pGLO. Label both tubes with your group's name. Place them in the foam tube rack. 2. Open the tubes and using a sterile transfer pipet, transfer 250 μl of transformation solution - CaCl₂ 3. Place the tubes on ice.

Evaluation of disinfectant/antiseptics

1. Test the chemical agents provided. 2. To the 5 ml of disinfectant, add 0.5 ml of the E. coli culture. Gently shake the tube to mix thoroughly. Note the time. 3. Divide a nutrient agar or tryptic soy agar plate into four sections with a marking pencil/pen. AT INTERVALS OF 2, 5, 10, AND 15 MINUTES, transfer one loop of the mixture to a section of the agar plate that has been labeled according to time. Also label the name of the organism and disinfectant tested.

Describe the disk diffusion method for testing antibiotic sensitivity of an organism

1. The entire surface of a plate of nutrient medium is swabbed with organism to be tested. 2. The handle of the dispenser is pushed down to place a number of disks on the medium. Use a flame-sterilized loop to gently tap the disks to the plate. 3. After 18 hours of incubation, the zones of inhibition (diameter) are measured in millimeters. 4. Refer to your photographic atlas. Record the organism, the antibiotic tested, the zone of inhibition (mm.), and whether the organism was resistant (R), intermediate (I), or susceptible (S). A handout will be provided for interpretation of your results.

E. coli transformation and antibiotic resistance - 7

10. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and using a new sterile pipette, add 250 μl of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipette for the other tube. Incubate the tubes for 10 minutes at room temperature. 11. Tap the closed tubes with your finger to mix. Using a new sterile pipette for each tube, pipette 100 μl of the transformation and control suspensions onto the appropriate plates.

E. coli transformation and antibiotic resistance - 8

12. Spread the suspensions evenly around the surface of the agar by rotating the plates gently in a figure 8 shape on the table. 13. Stack up your plates and tape them together. Put your group name and class period on the bottom of the stack and place the stack upside down in the 37°C incubator until the next day. 14. Next week examine your plates using the U.V. light.

Serial Dilution and Pour Plate Method - results - 2

1:100 plate: # colonies x 1 ml x 100 = _____ organisms/ml 1:1000 plate: # colonies x 1 ml x 1000 = ____ organisms/ml 1:10,000 plate: # colonies x 1 ml x 10,000 = ___ organisms/ml 1:100,000 plate: # colonies x 1 ml x 100,000 = ______ organisms/ml 1:10,000,000 plate: # colonies x 1 ml x 10,000,000 = __________ organisms/ml Final plate count: # organisms/ml on 1:100 plate __________ + # organisms/ml on 1:1000 plate +# organisms/ml on 1:10,000 plate __________ + # organisms/ml on 1:100,000 plate + # organisms/ml on 1:10,000,000 plate = _____________.

Serial Dilution and Pour Plate Method - 2

2. You will be given a 1:100 dilution of either raw milk or pasteurized milk from which you will make further serial dilutions as follows: a. Using a sterile 1 ml pipette, transfer 1 ml of the 1:100 milk sample into a water blank (9 ml water). This is a 1:1000 dilution. Label the tube. b. Using the same pipette, transfer 1 ml of the 1:1000 dilution to another water blank. This is a 1:10,000 dilution. Label the tube. c. Using the same pipette, transfer 1 ml of the 1:10,000 dilution to another water blank. This is a 1:100,000 dilution. Label the tube.

Serial Dilution and Pour Plate Method - 3

3. Take 4 sterile Petri dishes. Mark the bottom of each, respectively: 1:100, 1:1000, 1:10,000, 1:100,000.

Serial Dilution and Pour Plate Method - 4

4. Measure 1 ml of the highest milk dilution (1:100,000), and deliver it into the bottom of the Petri dish so marked.

E. coli transformation and antibiotic resistance - 2

4. Use a sterile loop to pick up a single colony of bacteria from your starter plate. Pick up the +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tube back in the tube rack in the ice. Using a new sterile loop, repeat for the -pGLO tube.

E. coli transformation and antibiotic resistance - 3

5. Have your instructor dispense the plasmid into the +pGLO tube. Mix the loopful into the cell suspension of the +pGLO tube. Close the tube and return it to the rack on ice. Also close the -pGLO tube. Do not add plasmid DNA to the -pGLO tube. Why not?

Serial Dilution and Pour Plate Method - 5

5. Using the same pipette, repeat step 4 for each diluted milk sample, in descending order of dilution.

Serial Dilution and Pour Plate Method - 6

6. If the tubes of agar are melted, remove them from the water bath, and testing both water and tubes with your hands, make certain the agar has cooled to 45oC. Wipe off tubes and sterilize lip of tube before adding agar.

E. coli transformation and antibiotic resistance - 4

6. Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the ice.

Serial Dilution and Pour Plate Method - 7

7. Pour each tube of cooled agar into one of the Petri dishes containing a milk dilution. Replace the top of the plate and gently rotate to assure distribution of the milk in the melted agar.

E. coli transformation and antibiotic resistance - 5

7. While the tubes are sitting on ice, label your four agar plates on the bottom (not the lid) as follows: Plate 1: LB/amp plate +pGLO; Plate 2: LB/amp/ara plate +pGLO Plate 3: LB/amp plate -pGLO Plate 4: LB plate - pGLO.

E. coli transformation and antibiotic resistance - 6

8. HEAT SHOCK: Using the foam rack as a holder, transfer both the (+) pGLO and (-) pGLO tubes into the water bath, set at 42 °C, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the warm water. When the 50 seconds are done, place both tubes back on ice. For the best transformation results, the change from the ice (0°C) to 42°C and then back to the ice must be rapid. 9. Incubate tubes on ice for 2 minutes.

Serial Dilution and Pour Plate Method - 8

8. When each poured plate is completely solidified, invert it.

Serial Dilution and Pour Plate Method - 9

9. Incubate all plates at 35oC for 24 to 48 hours

Enrichment Media

A basic media on which a majority of clinical specimen bacteria grow. It contains agar enriched with blood (usually sheep blood) and other nutrients required by pathogens.

HYDROLYSIS OF STARCH

Add LUGOL'S IODINE to plate. Note the location and appearance of the growth before adding the iodine. Examine the plate for clearing around the growth. Also, iodine reacts with starch to produce a black-purple color. If the starch has been hydrolyzed the iodine will remain brown.

CITRATE UTILIZATION TEST

Blue color development in any portion of the slant is considered positive

How does the pGLO plasmid work??? [TEXT FROM LAB BOOK]

Gene expression in all organisms is carefully regulated to allow adaptation to differing conditions and to prevent wasteful production of proteins. Bacterial genes encoding the enzymes needed to metabolize the simple sugar arabinose are a perfect example. A promoter region upstream of these genes acts as a molecular on/off switch that regulates their expression. The genes are activated only when arabinose is present in the environment. pGLO plasmid incorporates the arabinose promoter, but the genes involved in the breakdown of arabinose have been replaced with the jellyfish gene encoding GFP. When bacteria transformed with pGLO plasmid are grown in the presence of arabinose, the GFP gene switches on, causing the bacteria to express GFP and fluoresce brilliant green.

Differential Media

In addition to basic nutrients, ___ media contain one or more components, such as a particular carbohydrate, that can be used by some microbes and not others. If the microbe uses the component during the incubation period, a change occurs in an indicator, also included in the media.

METHYL RED VOGES PROSKAUER TEST (two tests in one)

Inoculate a tube of MR-VP broth and incubate for 24 hours at 35oC. When growth is apparent, add 5 drops of METHYL RED indicator to 5 ml of MR-VP broth culture. A pH BELOW 4.5 = red = positive; a pH ABOVE pH 4.5 = yellow to orange = negative

Selective Media

It contains one or more components that suppress the growth of some microbes and not others

pGLO plasmid ... THREE PARTS ....

The pGLO PLASMID is constructed with the jellyfish gene that encodes: 1. GFP GENE. Green Fluorescent Protein makes them glow. 2. BETA-LACTAMASE GENE. An antibiotic-resistance gene that encodes β-lactamase protein and 3. AraC. The AraC gene encoding a regulator protein that turns the GFP gene on and off. Bacteria transformed with pGLO are ampicillin resistance, and, when induced to express GFP, the bugs glow fluorescent green under UV light!

THE ACTIVITY OF DEAMINASE (phenylalanine deaminase)

a. Inoculate each of the two cultures on a separate slant of phenylalanine agar. b. Incubate the cultures at 35oC for 24 hours. c. Examine the tubes for heavy growth. If adequate, but a 3 drops of 10% FERRIC CHLORIDE solution down the surface of each slant. d. Observe for the development of a green color. e. Refer to your photographic atlas and record your observations

THE ACTIVITY OF COAGULASE

a. Inoculate tubes of rabbit plasma with the test organisms (3 loops) and leave a third tube uninoculated as a control. Cover the tubes loosely with parafilm. b. Label tubes. c. Incubate the tubes at 35o C for up to 24 hours. d. Observe for solidification.

THE ACTIVITY OF DEOXYRIBONUCLEASE (Dnase)

a. Make a mark on the bottom of the DNA plate; divide in half, so that two different organisms can be tested on the plate. b. Heavily inoculate one side and rub the growth in a circle about the size of a quarter. Repeat on the other side with the other organism. c. Incubate the plate at 35o C for 24 hours. d. Examine the plate for growth and for clearing around the growth. g. Refer to your photographic atlas and record your observat

THE ACTIVITY OF GELATINASE

a. Stab inoculate two nutrient gelatin tubes with the test organisms. Leave the third tube uninoculated as a control. b. Label the tubes. c. Incubate the tubes aerobically along with the uninoculated control tube at room temperature for up to one week. d. Examine the control tube. If the control tube is solid, the test can be read. If the control tube has become liquefied due to the temperature, all tubes must be refrigerated or otherwise cooled until the control is resolidified. e. Examine the tubes for gelatin liquefaction.

Stab Inoculation

a. Stab-inoculate in and out without moving the needle around in the medium; your stab should only go down about one-fourth of the way into the agar. c. Incubate the tubes aerobically with the uninoculated control at 35o c for 24 to 48 hours. d. Examine the tubes for formation of a black precipitate in the medium (H2S production) and cloudy growth throughout the tube (motility). e. Add four or five drops of KOVAC'S REAGENT to each tube. f. Observe for the formation of a red color in the reagent layer (indole production).

THE ACTIVITY OF UREASE

a. Streak inoculate slants with the test organisms (2 tubes), covering the entire agar surface with a heavy inoculum. b. Label tubes. c. Incubate aerobically with an uninoculated control at 35o c for 24 hours. d. Observe for a change from orange to pink.

THE ACTIVITY OF CATALASE

a. Transfer a large amount of growth from slant culture to a microscope slide. b. Apply 1 or 2 drops of hydrogen peroxide directly to the bacteria on the slide. c. Observe for bubbling, indicating a positive reaction.

SIMPLE CARBOHYDRATE (PHENOL RED) FERMENTATIONS:

an indicator dye that turns YELLOW in acidic solutions and RED in alkaline solutions....and there's a bubble involved if there is fermentation.

SULFUR-INDOLE-MOTILITY (SIM) MEDIUM - 3 tests

formation of a black precipitate in the medium (H2S production) and cloudy growth throughout the tube (motility). Add four or five drops of KOVAC'S REAGANT to each tube. Observe for the formation of a red color in the reagent layer (indole production).