Lab exam 2
What is chemically defined and give an example.
media is composed of exact amounts of chemically pure, specifically identified organic or inorganic components.
-glucose salt broth
-inorganic synthetic broth
What is complex media and give an example.
composed of organic materials that are not chemically pure and not specifically identified chemical components
-tryptic soy broth/agar
What is enriched media and give an example.
Why is it necessary for growing fastidious bacteria?
-composed of general purpose media that has something added to help fastidious bacteria (those that require specific nutrients in order to grow, "picky eaters") to grow better
-blood agar, nutrient broth with yeast extract
What is the relationship between the amount of bacteria in a culture and the absorbance?
-as the amount of bacteria increases, the amount of turbidity increases, therefore the absorbance is increased.
What is the relationship between the amount of bacteria in a culture and the transmittance?
-100% transmittance would be your reading with zero cells
Would heterotrophic organisms grow well in inorganic salt media? Why or why not?
-heterotrophic organisms require organic compound for their energy and carbon source.
-without any organic molecules in the media, the bacteria will have nothing for energy or carbon.
-the bacteria will not grow in this media
Why is complex media generally used to cultivate microorganisms?
-it has lots of nutrients which would be required for the growth of most microorganisms.
-for most microbes, this is enough for growth
Describe the minimum, optimal and maximum growth temperatures.
minimal-the lowest temperature at which a bacterial population can survive
optimal-the temperature at which bacteria thrive (greatest rate of metabolism and reproduction)
maximum-the highest temperature at which a bacterial population can survive
list the temperature range for Psychrophiles
0oC - 15oC (with a range of -15o C - 20oC)
List the temperature range for Mesophiles
20oC - 40oC (with a range of 10oC - 50oC)
List the temperature range for thermophiles
45oC - 80oC (with a range of 45oC - 80oC)
List the temperature range for hyperthermophiles
80oC and up
Differentiate between aerobes, anaerobes, facultatives and microaerophiles
obligate aerobes-have the ability to live in oxygen and require oxygen for metabolism
obligate anaerobes-are not able to live in oxygen and do not require oxygen for metabolism
facultative-have to preference, growth is seen in both aerobic and anaerobic environments
microaerophiles-can grow in oxygen, but only require a small amount for metabolism
Describe how the anaerobic chamber achieves an anaerobic environment.
anaerobic chambers take oxygen out of the air, combine it with hydrogen, and produce water
Describe the indicator that an anaerobic environment is present
condensation on the inside of the container
What is fermentation?
anaerobic metabolism in which organic molecules are the end electron receptor
What is being distinguished in the carbohydrate fermentation test?
Whether the bacteria ferment this sugar to an acid, or an acid and a gas
What is the indicator used in the carbohydrate fermentation test?
phenol red indicator
-yellow when acidic and hot pink when basic
What is the appearance of a positive result for a carbohydrate fermentation to an acid? To a gas?
-a bubble is present in the durham tube
What is the appearance of a negative result for carbohydrate fermentation to an acid? To a gas?
-no bubble in the durham tube
What sugar is in the MR-VP broth?
What is being distinguished in the MR and VP test?
MR-fermentation results in mixed acids
VP-glucose fermentation results in acetoin production
What reagents are used in the MR and VP test?
MR-Methyl Red Indicator
VP-alpha naphthol, 40% potassium hydroxide
What is the appearance of a positive result for the MR and VP tests:
both are red
What is the appearance of a negative result for the MR and VP tests
both are coppery or have no color change
Explain why you would expect no growth on the LB/amp/-DNA plate
the bacteria is sensitive to ampicillin and ampicillin is present in the media
Explain why you would expect isolated colonies to grow on the LB/amp/+DNA plate.
-not all bacteria obtained the plasmid containing the ampicillin resistant gene, therefore you get isolated colonies instead of a lawn of growth
Explain the purpose of the arabinose on the LB/amp/ara/+DNA plate
Arabinose is a sugar that acts as an inducer to the gfp gene. When arabinose is present it removes the inhibitor from the section of the plasmid containing the gfp gene and allows transcription and translation to take place. The gfp gene produces the green fluorescent protein and causes colonies of bacteria to have a green fluorescent glow when exposed to UV light
Explain why you would expect a lawn of growth on the LB/-DNA plate
There is no ampicillin present in the media to inhibit the growth of the bacteria on the plate
What is selective media?
media that prevent the growth of one type of bacteria so that only one kind of bacteria grows on the plate
What is differential media?
media that differentiates one kind of bacteria from another by a color change
Can a media be both selective and differential?
-enriched with 5% sheep's blood
-differential for type of hemolysis
-selective with 7.5% salt for salt tolerant bacteria (gram +)
-differential for mannitol fermentation
-indicator is Phenol Red
-pH <7 is yellow
-selective with Eosin and Methylene Blue for Gram- bacteria
-differential for lactose fermentation
-indicator is Eosin and Methylene Blue
-lots of acid accumulated=green metallic sheen
-selective with bile salts and crystal violet for Gram- bacteria
-differential for lactose fermentation
-indicator is neutral red
-pH <7 is pink precipitate
-selective with phenylethanol tolerance for gram+ bacteria
Starch hydrolysis (amylase)
-if starch is hydrolyzed by amylase (produced by bacteria), then no starch will be present around the bacteria
-reagent: iodine will detect starch in the media by producing a blue/black precipitate
-positive for starch hydrolysis: clear zone around the bacteria
-catalase (enzyme) breaks down hydrogen peroxide (substrate) into water and oxygen
-oxygen bubbles: effervescence is a positive result for catalase production
Phenylalanine deaminase (enzyme) will work on phenylalanine by removing the amine group to produce phenylpyruvic acid and ammonia
-Reagent: ferric chloride combined with phenylpyruvic acid to produce a green color = a positive for phenylalanine deaminase enzyme
SIM: Hydrogen Sulfide (Sulfur)
-amino acids that are metabolized, theosulfates, sulfates and sulfites are reduced by the bacteria to produce hydrogen sulfide
-presence is a black precipitate in medium = positive result for H2S production
- Tryptophanase is the enzyme that hydrolyzes Tryptophan. Indole is a product of Tryptophan hydrolysis.
-Reagent: Kovac’s reagent - Dark pink color = positive for Indole production
-medium is semi-solid and allows the bacteria to move throughout the medium if it is motile.
-Motile bacteria will make the entire medium look cloudy or opaque.
-Urease (enzyme) breaks down urea (substrate) producing ammonia.
-Indicator: phenol red;
-Positive for urease production = hot pink
-Tests for the ability of the bacteria to utilize the citrate in the media as the sole source of carbon with the citrase enzyme.
-Bacteria that do this then use the ammonium hydroxide and ammonium phosphate as a sole source of nitrogen. When they break down the ammonium phosphate and ammonium hydroxide, they release ammonia. This raises the pH to produce a basic environment.
-Bromthymol blue in the medium is the indicator which will turn Prussian blue (KU blue) when it is basic and yellow when it is acidic. So a positive result for Citrate Utilization = Prussian blue (KU blue) color. A Negative result for Citrate Utilization = forest green color (no change in the color)
Define transient microbiota
This is the bacteria that have been picked up throughout the
day as we are exposed to them. They have not (yet)
colonized the body and if pathogens, have not breached the
Define normal microbiota
Normal microbiota are bacteria that have colonized the body (not just the skin). They are normally commensals which do not harm us, or mutualists which help us while they help themselves
Name 4 locations where normal microbiota is present on the host
-Upper Respiratory Tract
-Lower Respiratory Tract
-Digestive Tract (Oral Cavity, Large Intestine and Rectum)
-Urinary Tract (External Urethra)
-Genital Tract (Vagina).
Describe the ecological relationship between most normal microbiota and the host
Most are commensals (win/null (no affect) or have a mutualism (win/win) relationship. Some can be pathogenic, if given the opportunity (opportunistic pathogens).
Explain how normal microbiota can sometimes be an opportunistic pathogen
If microbiota crosses physical barriers into normally sterile compartments of the body, or if the host is immuncompromised, the microbe can cause disease
Use "texture" to describe a colony on a plate.
can be smooth, rough, shiny and/or dull
Use "shapes" to describe a colony on a plate:
-punctiform: a pin point small colony
-filamentous: fuzzy or thin extensions coming from the center
-rhizoid: root-like extensions come from the center
-spindle: football shaped
Use "elevation" to describe a colony on a plate
-pulvinate: like a water droplet on a surface
-umbonate: raised area in the center
Use "margin" to describe a colony on a plate
-even: entire, smooth regular edge
-filamentous: thin filaments from the center
-lobulate: lobular extensions from center
-erose: serrated, like a blade
-curled: similar to a bullseye target
a chemical used to control microbes on an animate surface
chemicals used to control microbes on an inanimate surface
Give examples of antiseptics
Give examples of disinfectants
What are the factors that affect the efficiency of the disinfectants and antiseptics?
- concentration of the chemical
- length of exposure
- type of microbial population to be destroyed
- environmental conditions (temp & pH)
What does the zone of inhibition indicate about the bacteria's relationship to the chemical?
-the zone of inhibition is related to the sensitivity or resistance of the bacteria to an antibiotic
-the larger the zone, the more effective the antibiotic
a chemical produced by certain species of bacteria and fungi for the purpose of competing better with their competition. The term traditionally refers to the naturally acquired chemotherapeutic agents. However, so many of the naturally occurring antibiotics have been chemically changed in the laboratory that most of the antibiotics are truly “semi-synthetic” and not true antibiotics
a general term that describes any chemical that can be used in the treatment or prevention of disease (it thus includes cancer chemotherapeutic agents as well as infectious disease chemotherapeutic agents).
What is the minimum inhibitory concentration (MIC) and how is it determined?
-the most dilute concentration of antibiotics that are still able to kill microbes.
-Two methods are used – the MIC test uses several tubes of serially diluted antibiotics (1:2, 1:4, 1:8, etc…), the same concentration/amount of bacteria is added to each tube and incubated. The MIC is then determined by looking at the most dilute solution that is still effective against the bacteria (no growth in the tube).
-The second method is the E-Test in which strips with increasing dilutions of the antibiotic on the back side of the strip are placed on an inoculated plate. Wherever the zone of inhibition crosses the strip, that indicates the MIC.
What conditions must be controlled to make the MIC test repeatable and accurate?
- Temperature and Time of Inoculation: 37˚C for 24-48 hours
- Concentration of Bacteria: 0.5 McFarland Turbidity
- Standard Concentration of Antibiotics: Standardized discs with antibiotics
- Type of media: Mueller Hinton Agar
What are the modes of action for the antibiotics used in our lab.
-azithromycin: 50S protein synthesis
-PCN: cell wall
-ciprofloxacin: nucleic acid-DNA
-sulfamethoxazole X trimethroprim: folic acid metabolism
-tetracycline: 30S protein synthesis
Name the 5 genera of Enterobacteriaciae bacteria tested for in this unknown lab.
Why are biochemical tests required to identify bacteria?
The bacteria all are gram negative rods and without biochemical tests, they are difficult to distinguish.
Why are biochemical tests required to identify the genus of these bacteria?
The biochemical tests indicate a set of unique characteristics
How are fermentation tests used to identify bacteria?
The results of fermentation are unique to each genus and can help identify the microbe
What are the five I’s of the microbiology lab and how were they used in this lab?
- Inoculation – when the media was inoculated
- Isolation – when the streak plate was performed
- Incubation – when the bacteria was incubated after inoculation
- Inspection – when the gram stain was observed
- Identification – when the bacteria was identified using biochemical tests
What does ELISA mean?
Enzyme Linked Immunosorbent Assay
Why is it better to have and Enzyme-linked antibody rather than Substrate-linked antibodies in this test?
Enzymes are recycled and only one enzyme linked antibody would be enough to create a visible change in the well to indicate an antigen/antibody reaction had occurred. If only one Substrate-linked antibody was present, that would not be adequate to be able to create a visible change
Where did the primary antibody come from in an indirect ELISA test?
patient's serum (if present)
Why should the secondary antibody stick to the primary antibody in an indirect ELISA test?
It is an anti-IgG antibody and will attach to any antibodies still in the well.
Why is it necessary to switch tips or pipettes when removing serum from the wells of different rows (between patients’ serum or between different dilutions).
To prevent any false positives from mixing of patients’ serum.
Why is it necessary to wash between each step?
This is to wash away any antibodies or antigens that did not stick to the wells or to the known antibodies/antigens.
What type of diagnostic test is the Bactistaph Test?
What is the Bactistaph test testing for to identify Staph bacteria?
Protein A is a part of the Coagulase enzyme that helps determine if the bacteria is Staphylococcus aureus or not.
What type of diagnostic test is the Group A Strep Test?
It is a type of Direct ELISA test (Sandwich Capture).
How are Streptococcus bacteria classified?
The two main ways that they can be classified are their Lancefield group of Carbohydrate Chain (Group A, B, C, etc….), or by Hemolysis (Beta hemolytic, Alpha hemolytic).