Principles of Microbiology Laboratory Study Guide for Lab Exam 2

Why is the decolorizer step critical in Gram Staining?

It differentiates between Gram-positive and Gram-negative bacteria, based on their cell wall structure and allows the initial crystal violet stain to be washed away from Gram-negative bacteria.

In microscopy, what does using an oil immersion objective (100x) require?

The use of oil to increase refractive index.

What is EMB Agar?

A selective and differential medium used to isolate Gram-negative bacteria that inhibits Gram-positive bacteria and helps differentiate between lactose fermenters and non-fermenters.

What components make EMB plates selective and differential?

Dyes eosin Y and methylene blue, along with the substrate, lactose.

What organisms are inhibited, and which are able to grow on EMB plates?

Gram-positive organisms are inhibited, while Gram-negative bacteria such as E. coli and Enterobacter can grow (lactose fermenters)

What do the different results in EMB plates represent?

There are three results: lactose fermenters (purple colonies, aerogenes-type growth), non-fermenters (colorless colonies), and strong fermenters (metallic green sheen, coli-type growth).

What is Phenol-Red Sorbitol broth?

A differential medium used to identify coliforms and enteric bacteria that can ferment sorbitol. It contains phenol red as a pH indicator that changes color in response to acid production.

What organisms are inhibited, and which are allowed to grow in Phenol-Red Sorbitol?

The medium inhibits non-sorbitol fermenters like Salmonella and Shigella while allowing the growth of coliforms such as E. coli.

What indicates positive and negative results in Phenol Red Broth?

Positive results are indicated by a color change to yellow, showing acid production, while negative results remain red, indicating no fermentation.

What does the Durham tube detect in fermentation tests?

Gas production.

What is Motility Deep?

A semi-solid growth medium used to determine bacterial motility. It contains tetrazolium salt which turns red when reduced by motile bacteria.

What components make the Motility deep a differential medium?

The substrate, tryptophan, and the indicator, Kovak’s Reagent.

What organisms are inhibited, and which are allowed to grow in motility deep?

Non-motile organisms are inhibited, while motile bacteria are allowed to grow.

What does the motility deep detect?

the ability of bacteria to move around and the presence of tryptophan degradation.

What does the IMViC series of tests stand for?

Indole, Methyl Red, Voges-Proskauer, and Citrate tests.

What medium is used for Indole Production test and what enzyme is assessed?

The medium used is tryptone broth, and the enzyme assessed is tryptophanase.

What does the Methyl Red test indicate?

Mixed acid fermentation.

What makes the Methyl Red test differential?

It differentiates organisms based on their ability to perform mixed acid fermentation, producing stable acids that lower pH.

What does the Methyl Red test inhibit and promote?

It inhibits the growth of non-fermenters and promotes the differentiation of enteric bacteria based on their fermentation pathways.

What do the results of the Methyl Red test show?

A positive result indicates a low pH (red) due to the presence of stable acids, while a negative result shows a neutral pH (yellow-orange)

What does the Vogues-Proskauer test indicate?

2’,3’-butanediol fermentation in microorganisms

What does the Vogues-Proskauer test inhibit and promote?

It inhibits non-fermenters and promotes the detection of acetoin production in enteric bacteria.

What makes the Vogues-Proskauer test differential?

It differentiates between organisms that can and cannot ferment glucose, based on acetoin production.

What are the components of the Vogues-Proskauer test?

Barritt’s Reagents A and B and glucose

What does Simmons’ Citrate Slant test for?

the metabolism of citrate

What makes the Simmon’s Citrate Slant a differential medium?

The substrate, citrate, and indicator bromothymol blue allow for differentiation between organisms that can utilize citrate as their sole carbon source and those that cannot.

What does the Simmon’s Citrate slant inhibit and promote?

It inhibits the growth of Gram-positive organisms and promotes the growth of Gram-negative organisms, specifically enteric bacteria.

What do the results of the Simmons Citrate Slant indicate?

A green color indicates negative results for citrate utilization while a blue color indicates positive results for citrate utilization.

What are the reactions involved in nitrate reduction?

The reactions involved in nitrate reduction include the conversion of nitrate (NO3-) to nitrite (NO2-) and further reduction to nitrogen gas (N2) or ammonia (NH3). This process can take place through denitrification or nitrate respiration.

What does the term 'confirm the presumptive negative' refer to in nitrate reduction and how is it done using zinc dust?

To verify that nitrate was not reduced to nitrite, using zinc dust. Zinc dust reduces any unreduced nitrate to nitrite, leading to a red color change if nitrate was indeed present, thus confirming the presumptive negative result.

What is the purpose of the Oxidase Test?

To identify organisms that produce cytochrome c oxidase.

How can the Oxidase test clear up ambiguities created by ferric sulfide precipitation in TSI tubes?

The Oxidase test can differentiate between organisms that produce cytochrome c oxidase and those that do not, helping to clarify whether observed color changes in TSI tubes are due to hydrogen sulfide production or actual fermentation of carbohydrates.

What indicates a positive and negative result of an Oxidase test?

A positive result is indicated by a dark purple color on the filter paper within 30 seconds, while a negative result shows no color change.

What indicates a positive and negative result in the Phenylalanine slant?

A positive result is shown by a green coloration after the addition of ferric chloride, indicating the presence of phenylalanine deamination; a negative result shows no color change.

What makes the phenylalanine slant a differential medium?

The phenylalanine slant is considered a differential medium because it allows for the identification of organisms that can deaminate phenylalanine, producing phenylpyruvic acid, which reacts with ferric chloride to yield a color change.

What is the function of the Urease Test?

To determine if an organism can hydrolyze urea to ammonia.

What indicates a positive and negative result for the Urea broth?

A positive result is indicated by a color change to pink due to the increase in pH from ammonia production (+ result of urease activity) a negative result shows no color change.

How does the EC-MUG broth work?

The EC-MUG broth is used to identify the presence of Escherichia coli in water samples by detecting the enzyme beta-glucuronidase, which E. coli produces. When E. coli metabolizes MUG, a fluorescent compound is released, confirming its presence.

What does the results of the EC-MUG Broth indicate?

The results of the EC-MUG broth indicate the presence of E. coli in the sample, as positive reactions lead to fluorescence under UV light and gas in the Durham tube shows it’s positive for lactose fermentation.

What are the components of the EC-MUG broth?

The components of the EC-MUG broth include nutrients for growth, MUG (4-methylumbelliferyl-β-D-glucuronide) for detecting E. coli, and a Durham tube for gas collection. Also contains bile salts and lactose.

How is E. coli detected in the EC-MUG test?

By producing fluorescence under UV light after MUG hydrolysis.

What are the objectives of the watershed project?

Total coliform detection and enumeration, Escherichia coli detection and enumeration, Heterotrophic bacteria and fungi enumeration, Antibiotic resistivity patterns of E. coli isolates, DNA extraction from bacterial enrichment cultures, Polymerase chain reaction detection of antimicrobial resistant genes, Agarose gel electrophoresis analysis of PCR products. To assess water quality and microbial safety.

What is the scientific method?

A systematic approach used to investigate observations, acquire new knowledge, or correct and integrate previous knowledge, typically involving formulating hypotheses, conducting experiments, and analyzing data.

Collecting water samples from Triplett Creek step 1

USEPA-approved, sterile sample containers will be distributed to samplers prior to sampling to ensure contamination-free collection of water samples.

Collecting water samples from Triplett Creek step 2

samplers will safely wade to the middle of the stream and dip the sterile sample container to a depth of four inches with the open end of the container facing upstream.  If the stream exhibits a low flow, the sampler will push the mouth of the container upstream at this depth until the container is nearly full.  The opened mouth of the container will at all times be upstream of the sample collector, sampling apparatus, and any disturbed sediments.

Collecting water samples from Triplett Creek step 3

For every ten samples collected in the field, two samples and a field “blank” (sterile water poured into a sample container) will be collected from a single sample site.

Collecting water samples from Triplett Creek step 4

Sample containers will be sealed and marked with stream name, sample site, date, time, sample number, and name of sample collector(s).  A chain of custody form will be completed for sample(s) collected at each sampling site.

Collecting water samples from Triplett Creek step 5

The samples will be immediately chilled in an ice chest at a temperature of 1° to 4°C for transport back to the Laboratory of Microbiology or the Water Testing Laboratory at Morehead State University.  At the lab, the chain of custody forms will be completed by lab personal receiving the samples.  All samples will be processed for the assessment of bacteria density within six hours of collection.

What are the components of the medium used in the R-CARD?

it contains the chromogen BCIG (5-bromo-4-chloro-3-indolyl-β-D-glucuronide)

How does the R-card work?

E. coli expresses the enzyme beta-d-glucuronidase, which releases 5 bromo-4-chloro-3-indolyl (a blue pigment) which allows us to see the E. coli colonies

What is the IDEXX Quanti-tray/2000?

A 97-well tray that accommodates 100 mL of water sample

What does the IDEXX Quanti-tray/2000 test for and how does it do so?

Tests for total coliform (β-galactosidase+, lactose fermenting enterics that cleave the chromogen ONPG* to a yellow-colored end product) and Escherichia coli (β-D-glucuronidase+; cleaves the fluorogen MUG✦to a fluorescent end product).

What is the unit used to measure E. coli and Total Coliform in IDEXX trays

Most probable number (MPN)/100 mL

How do you get the MPN/100 ml of Total coliform and E. coli in IDEXX trays?

count the number of positive large wells and positive small wells and compare to the standard MPN table, then multiply that number by 10

What are positive results of IDEXX trays?

-TC - appear yellow under incandescent light

-E. coli - appear blue fluorescent under ultraviolet light

What are components of the medium Colilert-18?

Nutrient broth that allows for simultaneous coliform bacteria and E. coli detection.

What are R2A and RDM plate culture media used for?

To assess heterotrophic bacteria and fungi, respectively.

Kirby Bauer plate method step 1

Inoculate a Kirby-Bauer plate to confluency with 4-6-hr Mueller-Hinton broth culture of the test microbe.

Kirby Bauer plate method step 2

Apply 12 different antibiotic-impregnated disks onto the surface of the medium.

Kirby Bauer plate method step 3

Incubate for 18-20 hrs. at 35-37oC.

Kirby Bauer plate method step 4

Measure (in mm) the diameters of the zones of inhibition surrounding each disk.

Kirby Bauer plate method step 5

Compare to published standards (see handout entitled, “Antibiotic Sensitivity Analysis”) to determine:

-Resistant (R)

-Intermediate (I)

-Sensitive (S)

What principle does the Kirby-Bauer method demonstrate?

Antibiotic susceptibility testing.

What does polymerase chain reaction (PCR) do?

Amplifies specific DNA sequences.

What does the term 'specificity' mean in relation to PCR?

The ability to distinguish the target DNA from non-target sequences.

What does the term “sensitivity” mean in relation to PCR

The ability to detect even small amounts of target DNA sequences in a sample.

How does PCR take advantage of the properties of DNA polymerase?

PCR utilizes DNA polymerase's ability to synthesize new DNA strands complementary to the target sequence during the amplification process.

What components make up a Master Mix in PCR?

DNA polymerase, nucleotides, primers, and buffer.

What component goes into each PCR tube?

-Target DNA

-Sequence Specific forward and reverse primer

-Sterile RNase-free H2O

-PCR Master Mix

-microbes from the water sample TSA culture

What is an amplicon in the context of PCR?

The DNA fragment that is amplified.

What happens during the denaturation step in PCR?

The double-stranded DNA template is heated to separate it into two single strands.

What happens during annealing in PCR?

During annealing, primers bind to the complementary sequences on the target DNA strands, allowing for the initiation of DNA synthesis.

What happens during extension in PCR?

DNA polymerase synthesizes new DNA strands from the primers.

What is the purpose of agarose gel electrophoresis and how does it work?

To separate and analyze DNA fragments based on size, agarose gel electrophoresis uses an electric field to pull negatively charged DNA through a gel matrix, allowing smaller fragments to migrate faster than larger ones.

What is the hydrologic cycle?

It explains how water transitions between different states and locations in the environment, essential for supporting ecosystems and climates. Precipitation → Infiltration → Evaporation → Transpiration → Condensation

What is the human water cycle?

The human water cycle refers to how humans interact with, use, and manage water resources, including aspects like consumption, wastewater treatment, and the impact on local ecosystems. It highlights the importance of sustainable water practices for maintaining water availability for future generations.

What is the role of microorganisms in water treatment?

Microorganisms play a crucial role in water treatment by breaking down organic matter and pollutants through processes such as biodegradation and nutrient cycling, thereby cleaning wastewater and promoting ecological balance.

What is the role of microorganisms in wastewater treatment?

Microorganisms are essential in wastewater treatment as they decompose organic waste and contaminants through biological processes, leading to cleaner water and reducing environmental pollutants.

What is the primary concern in wastewater treatment?

The role of microorganisms in breaking down organic matter.

What standards does the EPA set for recreational waters?

Single grab sample count <240 E.coli CFU or MPN/100 mL. Geometric mean of 5 samples in 30 days <130 E. coli CFU or MPN/100 mL

What pathogenic bacterial organisms are known to contaminate water?

E. coli, Salmonella, Shigella, Vibrio cholerae.

What viral pathogens are threats in water?

Norovirus, Hepatitis A virus, Poliovirus.

What parasitic protozoans can be found in contaminated water.

Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica

What distinguishes a negative result in biochemical tests?

The absence of a color change or expected reaction.

What is the function of Kovac’s Reagent?

To detect indole production by forming a red ring upon reaction.

What does Bromothymol blue indicate in microbial tests?

pH changes, particularly in the presence of acid.

What is a major indicator of recreational water quality according to EPA standards?

E. coli counts in water samples.

How does temperature affect the thermocycle in PCR?

Different temperatures allow denaturation, annealing, and extension phases.

What does it mean if a culture shows no growth on selective media?

It indicates that the organisms inhibited by the selective agents are present.

What distinguishes selective media from differential media?

Selective media inhibits certain organisms, while differential media differentiates based on biochemical reactions.

What is unique about the selected sites in the Triplett Creek Watershed project?

They are chosen for their ecological significance and potential contamination sources.

What does the presence of total coliforms in water indicate?

Potential contamination by fecal matter and possible pathogens.