Define chemically defined and complex media. Give an example of each type of media.
Chemically defined media is composed of exact amounts of chemically pure, specifically identified organic or inorganic components. Examples include glucose salt broth or inorganic synthetic broth. Complex media is composed of organic materials that are no
What is enriched media and give an example. Why is it necessary for growing fastidious bacteria?
Enriched media is 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. Examples include Blood Agar, Nutrient Broth with Yeast Extra
What is the relationship between the amount of bacteria in a culture and the absorbance?
Directly Related. As the amount of bacteria increases, the amount of turbidity increases, therefore the absorbance is increased. Transmittance? Indirectly related to the amount of bacteria. 100% transmittance would be your reading with 0 cells.
Would heterotrophic organisms grow well in inorganic salt media? Why or why not?
Heterotrophic organisms require organic compounds for their energy source and their carbon source. Without any organic molecules in the media, the bacteria will have nothing for energy or a carbon source. Therefore, the bacteria will not grow in this medi
Why is complex media generally used to cultivate microorganisms?
Complex media has lots of nutrients (amino acids, sugars, vitamins and minerals) which would be required for growth of most microorganisms. For most microbes, this is enough for growth.
Psychrophiles
0-15�C
Mesophiles
20-40�C
Thermophiles
45- 80�C
Hyperthermophiles
80�C
have the ability to live in oxygen and require oxygen for metabolism
Obligate aerobes
are not able to live in oxygen and do not require oxygen for metabolism
Obligate anaerobes
can grow in oxygen, but only require a small amount for metabolism
Microaerophiles
have not preference - growth is seen in both aerobic and anaerobic environments
Facultative
Describe how the anaerobe chamber achieves an anaerobic environment.
Anaerobe chambers take oxygen out of the air, combine it with hydrogen, and produce water.
Describe the indicator that an anaerobic environment is present.
Indicator tablets are purple in oxygen and red in anaerobic environments.
Compare ionizing and non-ionizing radiation.
Ionizing radiation is so great that it can eject an electron from the atom. Ionizing radiation produces free radicals which damage DNA and completely break down DNA (rather than mutating). Non-ionizing radiation has less energy and is unable to eject an e
Give examples of ionizing and non-ionizing radiation.
Ionizing - Gamma and X-rays
Non-Ionizing - UV radiation
What is the germicidal range for electromagnetic radiation?
<300 nm (less than 300nm)
What is the germicidal range for Ultraviolet Radiation?
210-300nm
What the effect of UV light on DNA?
UV light causes Thymine Dimers to form (covalent bonds connecting the Thymine next to each other on a
strand of DNA. This inhibits the replication and transcription of that strand of DNA.
What is the appearance of Serratia marcescens that has been mutated by UV light?
S. marcescens that has been mutated by UV light does not produce the red pigment and therefore would have a cream color.
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 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 carbohydrate fermentation to an acid? To a gas?
Yellow; bubble in the durham tube (inverted tube inside the test tube.)
What is the appearance of a negative result for carbohydrate fermentation?
Red(not yellow)/No bubble
What sugar is in the MR-VP broth?
Glucose
What is being distinguished in the MR and VP tests?
MR - Fermentation results in Mixed
Acids; VP - Glucose Fermentation results in Acetoin
What reagents are used in the MR and VP tests
MR - Methyl Red Indicator; VP - Barritt's
A = alpha naphthol; Barritt's B = 40% Potassium Hydroxide (KOH)
What is the appearance of a positive result for the MR and VP tests?
Red (not coppery
color)
What is the appearance of a negative result for the MR and VP tests?
Coppery or no color
change (Not red)
Explain why you would expect no bacteria growth on an LB/amp - DNA plate.
Because the bacteria is sensitive to ampicillin and ampicillin is present, there would be no bacteria growing on the plate (any bacteria that may be growing must have obtained the ampicillin resistance gene.)
Explain why you would expect isolated bacteria to grow on an LB/amp plate +DNA plate.
Isolated growth occurs on this plate rather than a lawn of growth because not all bacteria obtained the plasmid which contains the ampicillin resistance gene. Not all bacteria present were transformed. Those that were transformed grew on the LB/amp plate
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 gr
Explain why you would expect a lawn of bacteria on the LB -DNA plate.
On the LB, DNA- plate, there is no plasmid containing the ampicillin resistance gene. However, there is also no ampicillin present. Therefore there is nothing to inhibit the growth of the bacteria on the plate and a lawn of bacteria will grow.
Define antiseptic and disinfectant. Which of the chemicals used were antiseptics? Disinfectants?
Antiseptic is a chemical used to control microbes on animate (living tissue) objects. Disinfectants are chemicals used to control microbes on inanimate (non-living) objects. Antiseptics used were - hydrogen peroxide, alcohol, iodine. Disinfectants were -
What are the factors that affect the efficiency of the disinfectants and antiseptics?
1. 2. 3. 4.
Concentration of the chemical, length of exposure, type of microbial population to be destroyed, and environmental conditions (temperature and 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 (the more sensitive or susceptible the bacteria is to the chemical).
Compare the terms antibiotic and chemotherapeutic agent.
Antibiotic is 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 occur
What is the minimum inhibitory concentration? How is it determined?
The minimum inhibitory concentration is 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 concentrat
What conditions must be controlled to make this test repeatable and accurate?
Temperature and Time of Inoculation - 37 ?C for 24 hours Concentration of Bacteria - 0.5 McFarland Turbidity Standard is used Concentration of Antibiotics- Standardized discs with antibiotics are available Type of media- Mueller Hinton Agar
What are the modes of action for the antibiotics used?
Most of the modes of action for the antibiotics used in our lab one of the targets discussed in chapter 12: Azithromycin (Protein synthesis), Cefoxatin (Cell Wall); Ciprofloxacin (Nucleic Acid); Sulfmethoxazole x Trimethroprim (Folic Acid/Metabolism) inhi
Define Transient Flora
This is the bacteria that have been picked up
through out the day as we are exposed to them. They have not (yet) colonized the body and if pathogens, have not beached the physical barriers.
Define Normal Flora
Normal flora (or microbiota or normal biota) 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.
Explain why soap is useful in handwashing.
Soap is a degerming agent, which means it will help to wash away microbes.
In terms of microbial control, how is soap useful?
It is weakly germicidal and useful as a degerming agent.
What is the mode of action of soap?
It targets the cell membrane and lyse the cell.
What chemical agent is usually combined with soap to make it "anti- bacterial"?
Phenols like triclosan or chlorohexidine are often combined with soap to make it anti-bacterial. Iodine is also often combined with the soap to make it anti-bacterial.
Wouldsoapbe"anti-bacterial"withoutthechemicalagent?Whatwould be the best term to describe soap without the chemical agent?
Soap is anti- bacterial without chemical agents, however weakly germicidal. The best term to describe soap without the chemical agent is "Surfactant".
What is selective media?
Media that prevents 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?
Yes
BAP
Differential
Enriched with 5% Sheep blood
MSA
Selective
7.5% salt
For salt tolerant bacteria
Differential - Mannitol fermentation - indicator - Phenol Red - pH <7 is yellow
EMB
Selective - with Eosin and Methylene Blue - for G- bacteria; Differential - Lactose fermentation - indicator - Eosin and Methylene Blue - lots of acid accumulated = Green Metallic Sheen
MAC
Selective - bile salts and crystal violet - for G- bacteria; Differential - Lactose fermentation - Indicator - Neutral Red - pH < 7 is pink precipitate
PEA
Selective - Phenylethanol tolerance - for G+ bacteria
Starch Hydrolysis (Amylase)
1. What are the enzymes? 2. What does this test distinguish? 3. What are the enzymes and substrates involved (if applicable)? 4. What sugar is fermented (if applicable)
5. What are the indicators and reagents? 6. Which are pres
If starch is hydrolyzed by amylase (produced by the bacteria), then
no starch will be present around the bacteria. Reagent- Iodine will detect starch in the medium by producing a blue-black precipitate. Positive for starch hydrolysis - clear zone around t
Catalase
1. What are the enzymes? 2. What does this test distinguish? 3. What are the enzymes and substrates involved (if applicable)? 4. What sugar is fermented (if applicable)
5. What are the indicators and reagents? 6. Which are present in the media an
Catalase(enzyme) breaks down hydrogen peroxide (substrate) into water and oxygen - oxygen bubbles = effervescence is a positive result for catalase production.
Phenylalanine Deaminase
1. What are the enzymes? 2. What does this test distinguish? 3. What are the enzymes and substrates involved (if applicable)? 4. What sugar is fermented (if applicable)
5. What are the indicators and reagents? 6. Which are present
by removing the amine group to produce phenylpyruvic acid and ammonia. Reagent - Ferric Chloride combines with phenylpyruvic acid to produce a green color.(Positive for Phenylalnine Deaminase)
SIM
1. What are the enzymes? 2. What does this test distinguish? 3. What are the enzymes and substrates involved (if applicable)? 4. What sugar is fermented (if applicable)
5. What are the indicators and reagents? 6. Which are present in the media and whi
hydrogensulfide-Aminoacidsthataremetabolized,Thiosulfates,sulfates,and sulfites are reduced by the bacteria produce hydrogen sulfide. Presence is a black precipitate in medium.
Indole - Tryptophanase is the enzyme that hydrolyzes Tryptophan. Indole is a p
Urease
1. What are the enzymes? 2. What does this test distinguish? 3. What are the enzymes and substrates involved (if applicable)? 4. What sugar is fermented (if applicable)
5. What are the indicators and reagents? 6. Which are present in the media and
urease(enzyme) breaks down urea(substrate) producing ammonia. Indicator - phenol red - Positive for urease production is a hot pink (fuchsia)
Citrate (Simmon's Citrate)
1. What are the enzymes? 2. What does this test distinguish? 3. What are the enzymes and substrates involved (if applicable)? 4. What sugar is fermented (if applicable)
5. What are the indicators and reagents? 6. Which are prese
Tests for the ability of the bacteria to utilize the citrate in the media as the sole source of carbon. 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
What does ELISA mean?
Enzyme Linked Immunosorbant 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
Where did the primary antibody come from?
Patient's serum (if it is present)
Why should the secondary antibody stick to the primary antibody?
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 .
Gram Reaction and Morphology of Staphylococcus?
Gram Positive staphylococcus bacteria are found on the skin.
What are the three species we worked with in class?
Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus.
Where are they found?
S. aureus
nose and throat
Where are they found?
S. epidermidis
skin
Where are they found?
S. saprophyticus
Urinary Tract
What diseases do they cause?
S. aureus?
wound infections, septicemia, food intoxication.
What diseases do they cause?
S. epidermidis
causes wound infections
What diseases do they cause?
S. saprophyticus
causes urinary tract infections
What are their respective results and appearance?
S. aureus
What are their respective results and appearance?
S. aureus
What are their respective results and appearance?
S. epidermidis
gamma hemolytic (no hemolysis), novabiocin sensitive (large zone of inhibition around novabiocin), no mannitol fermentation, catalase positive (bubbles), coagulase negative.
What are their respective results and appearance?
S. saprophyticus
alpha/gamma hemolytic (variable hemolysis), novabiocin resistant (no zone of inhibition around novabiocin disk), no mannitol fermentation (variable fermentation), catalase positive, coagulase negative (although slight agglutination on Bacti-staph test.)
Remember the following tests: 1. Hemolysis
2. Novabiocin (mode of action) 3. Mannitol Salt Agar 4. Catalase 5. Coagulase (enzyme, substrate, +/- test results)
notes
Gram Reaction and Morphology of Streptococcus
Gram positive streptococcus(chains or diplo of spheres)
How many Lancefield groups of Streptococci have been identified?
23
What is the primary pathogen in Group A strep?
Streptococcus pyogenes
Where are streptococcus found ?
throat
What does streptococcus cause?
Strep Throat
The Rapid Strep Test was used to identify which group of Streptococcus?
Group A
What is the basis of the Rapid Strep Test?
The membrane in the window is coated with
known antibodies to group A strep antigen and a control strip. When antigens have been removed from the swab containing the Group A Strep sample, they will flow with the control antigens up the membrane. The contr
What test would you use to distinguish between Staphylococcus and Streptococcus?
Catalase
Which is Catalase +? Which is Catalase -?
Staph is positive; Strep is negative.
Identification LAB
Why is a gram stain performed first in the identification process?
Knowing whether it is gram positive or negative and what the arrangement and shape of the bacteria are helps to narrow down the choices.
Why is a streak plate on Blood Agar performed?
Since Blood Agar is enriched, it helps the unknown bacteria grow better. You can also determine if the bacteria is alpha, beta, or gamma hemolytic which aids in the identification process as well.
What test can distinguish Staphylococcus from Streptococcus without doing a gram Stain?
Catalase can help differentiate Staph from strep. Staphylococcus are catalase positive.
Streptococcus are catalase negative.
Name the Enterobacteriaciae bacteria tested for in this unknown lab. Why are biochemical tests required to identify bacteria?
Escherichia, Enterococcus, Klebsiella, Proteus, and Salmonella. The bacteria all are gram negative rods and without biochemical tests, they are difficult to distinguish.
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 usin
How could Selective and Differential Media have helped in identification of the unknown bacteria?
Selective media would have helped determine whether the bacteria was gram positive/negative and whether salt tolerant or phenyl ethanol tolerant. Also it would help determine mannitol or lactose fermentation. This would help isolate the bacteria and start