Lab 7 Secondary Assays for Bacterial Characterization (1)

procedure/s in performing aseptic transfer of bacterial cultures in (include illustration) (3) agar plate to agar slant.

a.Describe what makes thioglycollate medium suitable for culturing anaerobes. What would the growth patterns of Clostridium sporogenes and Micrococcus luteus be in this medium?  b. In the Kligler test, why do we inoculate the surface of the agar slope and then stab into the butt of the slope? What does a pink coloured colony indicate when using MAC (MacConkey Agar)?

for E-coli- Describe results for Gram reaction, cell shape and arrangement (look up information) What color changes were observed for each test? Any zones of inhibition (for antibiotics only)?

Antibiotic sensitivity testing: The Kirby-Bauer. 1) What do the following terms mean? a. PPNG b. MRSA 2) How does the MIC test differ from the Kirby-Bauer test? 3) Were all of the conditions of a standardized Kirby-Bauer test met as you performed this assay? If not, which were not? 4) What is the significance of colonies that develop within otherwise clear zones of inhibition? If the laboratory report for one of your patients indicated colonies within the zone, what concerns would you have for your patient?

Please answer fast Dilution Problem. A culture of Staphylococcus is diluted as follows: (1) 20mL are added to 80mL of water. (2) 10uL from (1) are then added to 9.99mL of water. (3) A 10-2 dilution is made from tube # (2). (4) 100uL from (3) are plated for a pour plate and incubated.   Growth Problem.A culture with approximately 2x103 cells/mL were incubated. After 3 hours, the number of cells had increased to 3x105. a) How long was the generation time in minutes?b) How many generations have occurred

Considering microbial identification methods, to which category would the following method belong based on what it measures? * Performing a hydrogen peroxide breakdown assay to see if a microbe produces the enzyme catalase Serological O Biochemical Genotypic Phenotypic

procedure/s in performing aseptic transfer of bacterial cultures in (include illustration) (2) agar slant culture to agar slant

Subculture and Colony Morphology Descriptions Following identification of the Gram-negative isolate and Gram-positive isolate, you next subculture each onto fresh nutrient agar plates.  Briefly describe the subculture process in three or so sentences and what this allowed you to achieve; follow this with colony morphology descriptions of each.    Description:           Isolate A – Describe:    Colony morphology:    Medium & incubation temperature: Isolate B – Describe:    Colony morphology:    Medium & incubation temperature:

Вackground In order to determine whether a newly synthesized chemical might be a useful food preservative, the chemical was tested for its ability to inhibit bacterial growth. Control. 500 ml of cottage cheese was inoculated with 2 ml of a 24-hr culture of Pseudomonas aeruginosa and incubated at 25°C. Five hours after inoculation, a standard plate count showed there were 200 bacterial cells/ml in the cottage cheese. After 11 hours, 1600 cells/ml. After 21 hours, 50,000 cells/ml. After 27 hours, 400,000 cells/ml. Experiment. 500 ml of cottage cheese containing the preservative was inoculated with 2 ml of a 24-hr culture of P. aeruginosa. After 6 hours of incubation at 25°C, a standard plate count was performed. There were 700 bacterial cells/ml in the cottage cheese. After 14 hours, 11,000 cells/ml. After 20 hours, 90,000 cells/ml. After 28 hours, 1, 400, 000 cells/ml. Use the data and the semi-log graph paper to plot growth curves for the control and the experiment. Remember to label…

Match the chemical reaction to the visual effect of the product/biochemical test description     1. This assay's color change relies on an initial reduction reaction resulting in a black precipitate if the organisms is "positive"  2. The visual production of gas from this reaction indicates this organism can break down toxic byproducts of aerobic respiration. 3. This assay relies on a pH indicator (phenol red) for a color change. An organism that was "positive" for the certain enzyme in this assay, would result in a pink color change. 4. If an organism was incubated in simple broth containing a phenol red pH indicator and a sugar source, and the broth turned yellow following bacterial growth, that would be evidence of this reaction occurring.  5. The reaction representing this assay, works by producing a red colored product upon addition of a reagent, which tells us the organism possesses the enzyme required to break down a particular amino acid.

In aseptic technique, what will happen if the inoculated solid plated media is not inverted prior to incubation?

Special Media for Isolating Bacteria OBJECTIVES Because multiple methods and multiple media exist, you must be able to match the correct procedure to the desired microbe. For example, if bacterium B is salt-tolerant, a high concentration (>5%) of salt could be added to the culture medium. Physical conditions can also be used to select for a bacterium. If bacteri- After completing this exercise, you should be able to: 1. Differentiate selective from differential media. 2. Provide an application for enrichment and selec- tive media. um B is heat-resistant, the specimen could be heated before isolation. Dyes such as phenol red, eosin, or methylene blue are sometimes ineluded in differential BACKGROUND media. Products of bacterial metabolism can react with One of the major limitations of dilution techniques used to isolate bacteria is that organisms present in limited amounts may be diluted out on plates filled with dominant bacteria. For example, if the culture to be isolated has 1…

Background: The following results were obtained from a broth dilution test for microbial susceptibility. What is the minimum bactericidal concentration of this antibiotic and explain what you based your answer on?

Isolating a pure culture from a mix sample is typically done on solid agar but can be performed using a liquid media. How can you isolate a pure culture from a mixed culture in a liquid medium (broth, not agar)?

ANSWER THE FOLLOWING QUESTIONS REGARDING GEL ELECTROPHORESIS 1.why is it not advisable to move/touch the agarose gel in the process of hardening 2.what is the use or function of the TAE buffer that is poured or found in the gel box 3.how can you tell if agel is running 4.outline the process use in the preparation of agarose gel of 1.5 concentration 5.outline the processes/steps used in gel electrophoresis 6.name twoexamples of the dye used in gel electrophoresis 7.how do we prepare x1(concentration) TAE BUFFER form 50x TAE buffer

What is the result of this biochemical test in the gelatin deep?cWhich enzyme is this testing for? What tool is used to inoculate this medium?

Voges Proskauer test Does color of the medium change to red after both reagents were added and allowed to sit for 60 minutes? If yes, after about how many minutes before you can see red color?

Some Metabolic Activities of Bacteria A. Questions: 1. List 5 importance of using selected and differential culture media. 2. Mannitol Salt Agar is a differential or selective media? Explain why? 3. What enzyme and explain the principle on how Simmons citrate agar change in color for positive result? 4. What two groups of bacteria that can be differentiated with the catalase test?

Following electrophoresis of the two pGlo mini-preparation samples (both digested and undigested), the following agarose gel image was obtained. provide an accurate and detailed figure, describing the image and what it depicts.

Ex. 17: Chemotherapeutic Agents Using the Antimicrobial Susceptibility Table on pg 62 in the lab manual, determine whether not the bacteria plated in this Kirby-Bauer test are resistant (R), intermediate (I), or susceptible (S). The bacterial species tested was Escherichia coli (Gram negative). Ciprofloxacin Diameter = 20 Streptomycin Diameter 30 Carbenicillin Diameter 17 + Erythromycin Diameter = 23 Novobiocin Diameter = 0 bacterial growth = disk with antibiotic (see labels) = no growth

Identification of Gram-positive cocci: You have isolated a catalase-negative bacterium that doesn't grow well in 6.5% NaCl medium. Which test would you expect to give you a presumptive identification? (i.e. The first tests tell you the genus, which test would identify a species of that genus)   CAMP Test   Coagulase Tube Test   PYR Test   Novobiocin Sensitivity Test

Description 1. The fractions obtained from differential centrifugation are enriched but not pure. Explain how a greater degree of purification can be achieved using Density-gradient centrifugation and velocity centrifugation. 2. Explain equilibrium centrifugation.

A culture of Staphylococcus of unknown density is diluted as follows (letters represent each step): • 60 mL of a bacterial culture is diluted by adding 440 mL of water • 1000 microliters from (A) is added to 9 mL of water • Dilution (B) is used to prepare a 103 dilution in water • 100 ul from dilution (C) is plated to nutrient agar and incubated at 37°C Following incubation, colonies are uniformly distributed on the agar, and 102 colonies are counted on one half of the plate. 1. Express the total CFU/mL of the original culture in scientific notation. 2. If 10 ul is plated from dilution (C), how many colonies should grow on the plate? Edit View Insert Format Tools Table

in lab: . cath sample + antibiotic susceptible A. baumanii  _occurrence of conjugation and colonies seen ( WHITE)   question:   Why was there growth in sample (a) listed above?

Which biochemical test is this? Name the organism that will test positive for this. Was this an important test for gram negative bacteria? Which tube is positive and negative?

How would you identify this unknown bacteria using a flowchart and the bacteria below as a possible unknown? Bacillus cereus, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis and Lactococcus lactis. Create flow chart (dichotomues) using at least 3 other biochemical tests from the following list: Mannitol salt agar, Blood agar, Starch agar, Tributyrin Agar, Gelatin, Casein agar, Indole Production, MR-VP, Citrate, Hydrogen Sulfide test, Urease test, Nitrate reduction test, Catalase test, and Oxidase test.

microbial sensivity lab: in the procedure used to test bacterial growth against various temperatures (incubator, room, refrigerator, freezer), why should efforts be made to inoculate each tube with the same number of bacteria?

Cause-and-Effect Analysis: Given the conditions below, describe the effect on the staining procedure and explain briefly: 1. Excessive heat was applied during fixation 2. Low concentration of crystal violet used during gram staining 3. Excessive washing between steps 4. Insufficient decolorization 5. Excessive counterstaining

From figure  shows triplesugar iron agar. What biochemical characteristics does this figureillustrate? How could this medium be used to begin the identificationof the isolated bacteria in this chapter’s Case Study?

Considering microbial identification methods, to which category would the following method belong based on what it measures? * Examination of the shape and height of bacterial colonies growing on a plate Phenotypic Genotypic Serological Biochemical

Procedure Explant Preparation and Inoculation 1) The leaf explants were cut into squares (approximate size = 1cm x 1cm ). 2) The explants were soaked in 95% EtOH for 3 minutes with constant shaking. 3) EtOH was then decanted and 10% of Chlorox bleach was added together with a few drops of  Tween 20, with constant shaking. 4) The explants were rinsed with autoclaved distilled water for at least 5 times. 5) The explants were then left to dry in the laminar air flow cabinet. 6) The explants were cultured in the appropriate media (2 explants in basal MS without BAP and the other two in MS with BAP). 7) Three explants were placed on its upper surface down and the other three with lower surface down in both basal MS media with and without BAP. 8) All the culture jars (name, date and treatments employed etc.)were labelled and incubated them (in dark) in the growth chamber or culture room. 9) Explants were placed on the surface of the medium and the Petri dish were sealed with parafilm. 10)…

REMOVAL OF WATER FROM MICROBIAL CELLS USING FREEZE  DRYING METHOD. Before starting the freeze-drying process, the weight of the test tube, Wi which is 15.33g and test tube with microbial cell pellets, Ww which is 34.94g were weighed using an electronic balance. Plus, final weight of the beaker with test tube and cells is 70.35g. Then, the average amount of water removal and average cell yield were calculated by using the formulas given which were 9.81g and 0.5001. As a conclusion, there was 9.81g of water had been removed by using the technique freeze-drying for 24 hours by using a freeze dryer.    Question:  1.From this statement, please explain what process actually has happened during this freeze-drying method. 2.Make a clear conclusion that can made from this experiment.(explain briefly)

Please discuss how the Methyl Red-Vogues Proskauer test helps distinguish microbes from each other.

Drawings of the two bacterial cultures as observed under the 4 mm objective. Describe features observable under the high-power objective and their movement as seen in the hanging drop preparation

Topic: Preparation of Microbial Suspension A Pure colony is needed for inoculation in sterile water, a small portion of culture media was included. Effect:  Rationale:

What is the principle of freezing of cell lines and primary cells.Describe the general protocol for freezing cultured cells.In detail.