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Microbiology Lab Techniques and
Procedures
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Biol251L.Lab Practical Exam Review Part 2
General Microbio Lab (University of Nevada, Las Vegas)
It is important to have a thorough understanding of how these tests can build off one another to lead you to identification of an unknown. This skill may prove useful on the exam. It is also vital to see the practicality of these tests in the real world for diagnosis of disease. If an organism causes some human disease, it is probably useful to know that connection. You will have to perform aseptic technique and quadrant streak method in front of TA for points. You will also need to perform a gram stain to answer questions about a given organism.
Litmus Milk Test
Components and Purpose/Role: Litmus milk is an undefined medium that contains skim milk and azolitmin (pH indicator and oxidation/reduction indicator). Skim milk provides the nutrients needed for growth, lactose for fermentation, and casein a protein.
Azolitmin (also called litmus) is pink at pH 4.5 and blue at pH 8.3, but between these it will be a shade of light purple.
Four Basic Reactions:
Lactose Fermentation: makes the medium more acidic; lactose is hydrolyzed by beta-galactosidase into glucose and galactose; excess acid production can lead to precipitation of proteins and form an acid clot; gas production can lead to fissures/cracks in clot and in extreme cases can cause stormy fermentation. Reduction of Litmus: white color in the lower portion. Casein Coagulation: proteolytic enzymes in the bacteria can digest casein and coagulate the milk producing a curd.
Casein Hydrolysis: only partial digestion of casein leads to the production of ammonia which raises the pH (alkaline); full digestion will yield a clear fluid.
Genera Identified:
Used to differentiate between members of Clostridium and also the family of Enterobacteriaceae from other gram-negative rods since members of this family can reduce litmus.
Also lactic-acid bacteria can be identified like Lactococcus, Lactobacillus, Enterococcus, and Streptococcus.
Possible Results:
1= alkaline reaction 2= uninoculated control (pH around 6.8) 3= alkaline reaction 4= digestion reaction 5= acid clot, gas production, and litmus reduction 6= acid reaction 7= acid reaction, curd production
MR/VP Test
Broth Composition and Purpose/Role: MR-VP broth is a combination medium used to detect organisms capable of glucose fermentation that generate either acid end products or 2,3-butanediol. The broth contains peptone, glucose, and a phosphate buffer.
Peptone provides protein (also source of nitrogen) and glucose supplies a fermentable carbohydrate, while the potassium phosphate buffer serves to resist pH change in the medium.
Reagents Added:
MR: methyl red indicator dye.
VP: alpha-naphthol and KOH/creatine.
Methyl Red
VP stands for Voges-Proskauer.
Reactions Tested:
The MR test detects organisms capable of mixed acid fermentation, which will overcome the phosphate buffer and lower the pH (lactic acid, formic acid, propionic acid, succinic acid, and ethanol).
The VP test detects organisms capable of fermentation of glucose, but quickly convert their acid products into acetoin, which can be reduced to 2,3-Butanediol (little amounts of above acids but large amount of butanediol and ethanol).
Color Change Interpretation:
Starch Hydrolysis Test
Starch Structure:
Starch is a polysaccharide of α-D-glucose subunits that exists in two forms, amylose, which is linear and amylopectin, which is branched.
Enzymes Tested:
Two enzymes are necessary to hydrolyze starch to be of metabolic value to bacteria: α-Amylase and oligo-1,6-glucosidase.
Medium Components:
To test if an organism contains these two enzymes, a starch agar plate containing beef extract, starch, and agar is used.
Iodine Addition:
Once the plate has been incubated allowing bacterial growth and possibly starch hydrolysis, iodine is added to the plate.
Interpretation:
If an organism is capable, there will be a clearance around the bacteria. If there is a solid brown color with no clearance, no hydrolysis has occurred.
Urease Test
Purpose:
To identify the genus Proteus associated with UTI from other non- lactose fermenting enteric bacteria like Shigella and Salmonella.
Enzyme Tested:
Urea is the primary component of nitrogenous waste in urine from many animals including mammals. It can be hydrolyzed down to ammonia and carbon dioxide by the enzyme urease in bacteria.
End Products:
Carbon dioxide, ammonia, and H2O.
Urea Broth Composition:
Urea broth contains yeast extract (nutrient source), urea, and phenol red (pH indicator) among a few other ingredients.
pH Indicator:
Phenol red is yellow or orange at a pH below 8.4. However, if urea is hydrolyzed to release ammonia, the pH will rise leading the broth to change to a pink color.
Rapid vs. Slow Positive:
If the broth turns bright pink after an incubation time of 24 hours, it is considered a rapid urease-positive organism.
If the organism requires a longer incubation time up to 6 days, it is considered a slow urease-positive organism.
Genera Identified:
This test can distinguish Proteus from Salmonella and Shigella.
Bacitracin/Novobiocin Susceptibility Test
Bacitracin:
Bacitracin is an antibiotic produced by Bacillus licheniformis that inhibits bacterial cell wall synthesis. It is only effective on bacteria that have a cell wall in the process of actively growing.
Novobiocin:
Novobiocin is an antibiotic produced by Streptomyces niveus and it interferes with an enzyme necessary for DNA replication.
Purpose:
To see which organisms are susceptible to certain antibiotics.
Interpretation:
When the bacitracin disk is placed on a bacterial lawn, there will be a zone of clearance 10 mm or greater around the disk if the bacteria is susceptible.
A clearing of 16mm or more around the novobiocin disk shows susceptibility of the bacteria.
Genera Identified:
B: this test distinguishes Micrococcus (susceptible) from Staphylococcus (resistant).
N: this test is used to identify coagulase-negative Staphylococcus. S. saprophyticus is resistant.
Antibiotic Producers:
Bacitracin is produced by Bacillus licheniformis. Novobiocin is produced by Streptomyces niveus.
Bound coagulase (clumping factor) is attached to the bacterial cell wall and reacts directly with fibrinogen in plasma, which precipitates causing the cells to clump together in a mass.
Genera Identified and Clinical Applications:
Test to distinguish Staphylococcus aureus from other Gram-Positive cocci.
Staphylococcus aureus produces coagulase enzyme, which helps explain its high resistance to normal immune response and antimicrobials.
Interpretation:
+: firm clot -: liquid (S. epidermidis)
Motility Test
Purpose:
To detect motility.
Inoculation Method:
It is inoculated with a needle and straight stab through the middle.
Agar Concentration:
The agar concentration is reduced from 1.5% to 0.4% to maintain form of medium, but allow bacterial movement.
Interpretation:
If there is growth radiating from the central line of the stab, the bacteria is motile.
Fermentation can occur in the medium leading to cracks and cloudy growth, however this is not considered true motility (Klebsiella).
Genera Identified:
This is an important characteristic in Enterobacteriaceae. P. mirabilis is positive while S. epidermidis is not.
SIM Medium
Characteristics Tested:
Sulfur reduction, indole production, and motility.
Medium Composition and Role:
The SIM Medium is a multi-purpose test/semi-solid medium that contains casein and animal tissue (a rich source of amino acids, iron- containing compounds, and sodium thiosulfate-sulfur).
Sulfur Reduction:
Bacteria can reduce sulfur in two ways: cysteine desulfurase can hydrolyze cysteine to pyruvate and hydrogen sulfide (putrefaction), and thiosulfate reductase can reduce sulfate to hydrogen sulfide (anaerobic respiration).
The hydrogen sulfide gas can then react with the ferrous iron in the SIM medium generating ferrous sulfide, which forms a black precipitate (+).
Indole Production:
Kovac's reagent is added to test for indole production.
Positive test has a cherry red rosindole band at the top, and if a bacterium produces tryptophanase then it can hydrolyze tryptophan to indole, pyruvic acid, and ammonia.
Motility Observation:
You cannot see motility results if sulfur has been reduced in the medium.
Disinfectant Effectiveness
Definition of Disinfectant
A substance or other agent that destroys harmful microorganisms; an antiseptic. Antimicrobial substances that are designed to reduce the number of pathogens on a surface (non-living). The ones that can be used on living tissue.
Zone of Inhibition
What is the zone of inhibition and how is it calculated? A clearing around the antibiotic disks that show susceptibility or resistance, measured in diameter (mm). The zone of inhibition's edge does not indicate the limit of diffusion into the agar, it does not extend because the disks are at their minimal inhibitory concentration (MIC) making it not as powerful.
pGLO LB/Amp plate = white colonies that do not glow under UV light pGLO LB/Amp plate = No bacterial growth pGLO LB plate = even lawn of bacterial growth
Marker Genes
Resistance plasmid, encode genes for resistance to antibiotics or other growth inhibitors. Marker Genes are associated with an easily recognizable phenotype (such as antibiotic resistance or bacterial colony color), useful for selecting transformed cells from non- transformed cells.
Transformation Efficiency
Calculate transformation efficiency. Indicates the number of antibiotic resistant colonies per μg of plasmid DNA. TE = Total number of cells growing on the agar plate (LB/Amp/Ara) / Amount (μg) of pGLO DNA spread on agar plate Total amount of pGLO DNA used in the experiment = Concentration of DNA (μg/μl) x volume used (μl) Fraction of DNA used = Volume Spread on LB/Amp/Ara plate (100 μl) / Total Volume in +pGLO tube (510 μl) = 0.2 Amount (μg) of pGLO DNA spread on agar plate = Total amount (μg) of pGLO used in this experiment (0.8 μg) x fraction spread on the LB/Amp/Ara plate (0.2) = 0.16 μg Example 1: Total # of colonies counted on LB/Amp/Ara plate = 160 colonies TE = 160/0.16 μg = 1000 transformants/μg = 103 CFUs/ μg Example 2: Total # of colonies counted on LB/Amp/Ara plate = 250 colonies TE = 250/0.16 μg = 1562.5 transformants/μg = 1.5625 x 103 CFUs/μg
Plasmid Mini-Prep
Basic Steps of Plasmid Mini-Prep Procedure
Lysis of the cells to release DNA (SDS-NaOH) Removal of RNA (RNase solution) Removal of Proteins (ammonium acetate) Concentrate/De-salt plasmid DNA (Isopropanol precipitation, ethanol wash)
Explanation of Mini-Prep Reagents
GTE (Glucose-Tris-EDTA): the Tris buffers the cells at pH 7.9, while the EDTA chelates the divalent cations into the cell membrane and weakens the cell envelope. SDS-NaOH: lyses bacterial cells by dissolving the lipids in the cell membrane, and denatures some proteins, anionic detergent. NaOH: denatures plasmid and chromosomal DNA into single strands, plasmid DNA remains intact. KOAc (potassium acetate/acetic acid): acetic acid neutralizes pH to help plasmid DNA strands re-anneal, while the potassium acetate precipitates the SDS, proteins, and lipids trapping the renaturing
chromosomal DNA in this clump. The plasmid DNA and RNA remain in solution. Isopropanol: precipitates nucleic acids first and then slowly the proteins as well (time limit of 2 minutes is crucial here or the nucleic acids will be contaminated with proteins, proteases, etc.) Ethanol wash: remove remaining SDS-lipid/protein precipitate and salts. Tris/EDTA: Tris buffers the DNA solution. EDTA protects DNA from degradation by DNase by binding divalent cations that are necessary cofactors for DNase activity.
Agarose Gel Electrophoresis
Gel Preparation
This technique is used to separate DNA based on size. First, we must create the gel mold, which can be done with different percentages of agarose. Depending on the target DNA size, we can use a higher concentration of agarose (for smaller fragments-0.2 to 1kb) or a lower concentration of agarose (for larger fragments-5 to 10 kb). We will be using a 1% agarose gel because the pGLO plasmid is about 5kb. We will use 30-50 ml of agarose to fill the 7cm by 10 cm gel tray.
Calculating Agarose Amount
To calculate the amount of agarose needed to make 50 ml of a 1% gel: 1/100 = X /50 = 0.5 g of agarose (this should be weighed out and added to a flask).
Gel Staining and Visualization
Ethidium Bromide is used as an intercalating agent that is used as a fluorescent tag to view DNA bands under UV light (carcinogenic). The buffer used is TBE (Tris/boric acid/EDTA). A common loading dye is bromophenol blue or xylene cyanol. Bromophenol blue migrates slower and is best to be used for larger DNA fragments.
Gel Electrophoresis
It is important that the first well is loaded with a 1kb DNA ladder, which is used as a molecular-weight marker that helps compare DNA fragment size to known fragment bands. It has 13 ds blunt end fragments. The 1000bp and 3000bp bands will have a higher intensity on this gel compared to others and should serve as reference points. This tray is placed in an electric field, which will move the plasmid samples and DNA ladder through the gel from cathode (negative, black) to anode (positive, red). Larger fragments will move slower through the gel, whereas smaller fragments will move much quicker.
