Microlab midterm from others
Understand the basic principles behind a spectrophotometer and how it can be used to determine the concentration of bacteria in a culture.
A spectrophotometer is an instrument used for measuring the effective transmission of monochromatic light through a sample liquid. A beam of light is passed through a suspension and the amount of light transmitted by suspension is measured. The amount of light transmitted is inversely proportional to the amount of light absorbing or scattering particles in the solution. Can be used to determine concentration - Beer Lambert Law of Solutes
Urea:
An organic product of protein metabolism. Can be broken down by the urease enzyme to yield ammonia and carbamate.
Understand the reason for continually subculturing an unknown to a BHI slant.
BHI slant is your working stock culture. You will need to subculture a new slant each lab period as it is vital to have a fresh culture for setting up various biochemical tests.
Understand the Beer-Lambert Law of Solutes and how it is applied to determine bacteria concentration, as well as when it cannot be applied.
Beer Lambert Law - there is a straight line relationship between absorbance (also known as optical density) of a solution and the concentration of a solute. The optical density is a function of the original intensity of the light beam and the intensity of the beam passing through the solution. At very high or low solute concentration the relationship will no longer be linear. A bacterial suspension can also be considered the solute because of its light scattering properties. Therefore a linear relationship exists between its optical density and the number of cells per milliliter. The optical density does not directly indicate the number of cells in the population but only the light scattered by the population. To obtain an approximation of population size of a bacterial culture one must first determine the correct wavelength for the cells and then the OD values must be calibrated to a viable cell count to express microorganisms as CFU/ml/
Know the steps used in appropriate use of the spectrophotometer, and the reasons behind each step.
Before a sample can be measured the instrument must be set to zero with a blank. A blank has all the ingredients of the sample minus the aspect of the sample that you are trying to measure. A spectrophotometer contains a source of white light and an optical system which separates this light into its component wavelengths, collectively called its spectrum. The wavelength passes through the sample and strikes a photo-sensitive vacuum tube. The resulting electronic signal is amplified and displayed on the meter. 1) Adjust wavelength control, 2) blank with water, 3) adjust knob to zero absorbance, or 100% transmittance, 4) insert sample 5) close cover
Be able to determine CFU/ml from plate counts/dilution information, and how to properly report out that information according to microbiological standards. Ex 1: after plating 0.1 ml of a 10^-5 dilution, 158 colonies grow. What is the CFU / ml?
Calculating CFU / ml CFU / ml = (colony count) / (tube dilution * volume plated) CFU / ml = (158) / (10^-5 * 0.1) = 158 * 10^6 For more than one CFU/ml use an average. Note: if you've determined the final dilution for the plate, then volume plated has already been factored in. Just divide colony count by final dilution.
Understand the purpose for how to correctly interpret the catalase test. How is it performed? What are the possible reactions that could occur? What does each reaction mean?
Catalase which breaks down hydrogen peroxide to water and oxygen, the test is used to separate the staphylococci from the streptococci, enterococci, and lactococci. Streptococci, enterococci and lactococci are aerotolerant anaerobes that use an alternate enzyme, peroxidase to break down H2O2. Exoenzymes are enzymes that are released to the outside of the cell and are commonly used by gram positive bacteria. Place one drop of H2O2 on an isolated colony. Immediate bubbling indicates a positive test for the catalase enzyme due to the enzymatic conversion of hydrogen peroxide to oxygen and water. No bubbling indicates an organism that lacks catalase enzyme.
What types of microbes are present and where would they naturally be found? (i.e. in the environment)
Cellulose is degraded to fermentation products by the anaerobic genus Clostridium. Desulfovibrio uses the fermentation products as an energy source and sulfate as a final electron acceptor and produces hydrogen sulfide that will produce a black color. H2S diffuses to the aerobic zone where it is used by chemolithotrophs such as Beggiotoa and Thiobacillus as an energy source. Thiobacillus can also oxidize ferrous ion to ferric ion and produce ferric oxide. Green algae and cyanobacteria undergo oxygenic photosynthesis producing oxygen for aerobic organisms, while purple and green photosynthetic bacteria undergo anoxygenic photosynthesis. There are just some of the many reactions that result in nutrient cycling in these columns. The Winogradsky columns you will observe were started more than 20 years ago and hey have been provided with nothing except water and sunlight.
Determining dilution. Example: 1 ml of culture was transferred to 99 ml of diluent. What is the dilution? Example #2: 1 ml of culture is transferred to 9.0 ml diluent. From this initial dilution, four serial 10-fold dilutions are made. Determine the final dilution. (to arrive at a final dilution, multiply successive dilutions together).
Determining dilution: All final answers of bacterial concentrations should be written with two significant figures, 1 decimal place, in proper scientific notation (1.5 * 10^8). Dilution = (volume transfered)/(volume transfered+diluent volume) = vol transferred / total volume first: (1 ml) / (1 ml + 99 ml) = 1 / 100 = 0.01 or 10^-2 second: (1 ml)/((9+1) ml) (1 ml)/((9+1) ml) (1 ml)/((9+1) ml) (1 ml)/((9+1) ml) (1 ml)/((9+1) ml) (1/10) (1/10)*(1/10) (1/10)*(1/10)*(1/10) (1/10)* (1/10)*(1/10)*(1/10) (1/10)^5 = (10^-5)
Oxygen level impact
E. coli is a facultative anaerobe, so it can grow with or without oxygen, although it produces more ATP by aerobic respiration. Therefore, it will grow with or without the shaking, but growth rates are higher for the treatments that involved shaking (O2). Physiologically, higher concentrations of oxygen promote higher leakage of reactive oxygen species which may impair growth.
Know how a growth curve is normally determined for a bacterial population.
Growth curves are typically determined using the viable plate method. This method employs spreading a diluted sample of bacteria over a solid medium and determining the number of colonies that arise. The number of viable microorganisms in the sample is calculated from the number of colonies formed multiplied by the dilution factor (inverse of the dilution).
Ornithine decarboxylation:
If the tube is purple grey or purple then ornithine has been decarboxylated and the ornithine decarboxlase enzyme is produced. If the tube is yellow or yellow white acid products have been produced due to glucose fermentation but ornithine has not been decarboxylated and the organism lacks the enzyme.
What is in the Winogradsky column
In these columns a core of soil or sediment is mixed with Na2SO4, Na2CO3 and cellulose plus water. It is incubated in the light to allow the growth of photosynthetic organisms. A series of reactions occur in the column as they mature and particular microbial communities develop in specific microenvironments within the column. The height of the column allows an aerobic zone at the surface with microaerophilic and anaerobic zones below. Specific microbial populations grow at different levels because of different environmental conditions.
Understand the purpose for and how to correctly interpret mannitol salt agar. What type of medium is this? What is the purpose of each ingredient? What are the possible reactions that could occur? What does each reaction mean?
MSA is commonly used to isolate and differentiate the staphylocci and closely related organisms. The medium is selective due to high salt content which inhibits gram negative bacteria and many gram positives. Salt tolerant bacteria that can ferment mannitol will produce acids, causing the typically pinkish medium to change to yellow, due to the presence of the pH indicator phenol red.
MR test
Methyl red dye indicator that changes color at a pH below 4.5 which occurs when bacteria produce acid end products from fermentation. The development of a red color indicates the presence of mixed acid fermentation products. If no acid is produced it will remain yellow.
Know how to determine molarity (M) of a sample, given the molecular weight and concentration. IE: molecular weight of methylene blue is 320 g/mol and the concentration of the original sample is 0.005 mg/ml. Determine the molarity.
Molarity = mol / L (1 mol / 320 g ) * ( 0.005 g / L ) = mol / L
Understand the use of motility agar and how to interpret it.
Motility agar is an excellent way to determine the motility of most bacteria. It contains one-fifth the agar normally found in solid medium and also contains gelatin. This makes the medium semi-solid and allows the bacteria to move out into the medium if they are motile.
Understand the purpose for and how to correctly interpret nitrate broth medium. What type of medium is this? What is the purpose of each ingredient? What are the possible reactions that could occur? What does each reaction mean?
Nitrate broth is used to determine a bacterium's ability to reduce nitrate using the process of anaerobic respiration. Nitrite is detected by the addition of sulfanilic acid (Nitrate A) and dimethyl-alpha-naphthylamine (Nitrate B) to inoculated nitrate broth while nitrogen gas is detected by the addition of powdered zinc to inoculated nitrate broth (after reagents A and B have been added) Add 4 drops A then 4 drops of B, in the presence of nitrite these reagents cause the culture to turn red, indicating that nitrate reduction has occurred. See flow chart
Know what plates to use for calculation of CFU/ml and why.
Not all bacterial cells produce colonies as some bacteria tend to clump or aggregate. For this reason results are reported as colony forming units (CFU/ml) of a bacterial culture. Only plates with 25-250 colonies are used in determination of a bacterial concentration. Counts above are TNTC and cannot be used because it is impossible to tell if colonies are truly separate. Plates with less than 25 colonies have been shown experimentally to have very poor accuracy in predicting bacterial concentration.
What is the purpose of a Winogradsky column? What does it represent? What types of environments is it used to study?
Only 1% of the microorganisms on Earth have been cultured. There are ways to get these organisms to grow even if it is not in a medium or culture. Another way to grow these types of microorganisms are with a Winogradsky column. These columns are named after the Russian microbiologist, Sergei Winogradskky, and are a model ecosystem used to study soil and sediment microbiology. Most microorganisms found in the columns cannot be grown on ordinary laboratory medium, they are unculturable.
Be able to determine CFU/ml from appropriate plates from a growth curve experiment.
Only statistically valid if the sample yields between 25-250 colonies. EX: *25-250 Dilution (10^-6), #colonies: TNTC, 254, 240, 268, 251, TNTC Dilution (10^-7), #colonies: 0, TNTC, 27, 31, 23, 32 (24 + 27 + 31 + 32) / (4) * (10^7) = 2.9*10^8 CFU/ml
Understand the purpose for and how to correctly interpret OF medium. What type of medium is this? What is the purpose of each ingredient? What are the possible reactions that could occur? What does each reaction mean?
Oxidative-fermentative medium is used to determine whether an organism metabolizes carbohydrate strictly by oxidative respiration, by fermentation, or both. The carbohydrate substrate (typically glucose) is added to a basal media which also contains peptone. Bromthymol blue is the indicator dye. It is green at a pH near neutrality, yellow at an acid pH an blue at an alkaline pH. Bromthymol blue also inhibits the growth of most gram positive organisms, making the medium selective and differential. If acid is produced (green yellow) in both the open and closed tubes it is oxidative and fermentative If acid is produced in only the open tube the organism is oxidative and grows by respiration If acid is only produced in the closed tube the organism is fermentative and anaerobic.
Know how a Petrifilm is set up and how it differs from a typical agar plate.
Petrifilm contains a dehydrated medium that needs to be inoculated with one full milliliter of a sample to rehydrate the medium. There are several different varieties of Petrifilm available, each designed to grow a particular organism or group of organisms. We will be using Aerobic Plate Count (APC) petrifilm, designed as an all purpose medium for bacteria, or Coliform Count Petrifilm, which is specific for the growth of coliforms, such as E. coli.
Know how to determine generation time from a Time vs. Cell Number graph.
Select 2 points in the exponential growth phase that represent a doubling in the population size. Draw horizontal lines from each point to intersect with the best fit line. Draw vertical lines down to intersect with the x-axis. Determine the difference between two points.
ornithine decarboxylase info
Some bacteria produce enzymes which can remove CO2 from an amino acid in a decarboxylase reaction. Decarboxylases are inducible enzymes which are produced under the following conditions: 1) the organisms must have the gene for the decarboxylase 2) the environment must be acidic 3) the amino acid specific for the decarboxylase must be present.
chemoorganoheterotrophs
Some bacteria require organic forms of carbon for both energy and carbon
photoorganoheterotrophs
Some bacteria use light energy and organic forms of carbon
phototrophs
Some bacteria use light energy in photosynthesis
Understand the purpose for and how to correctly interpret the MR-VP tests. What type of medium is it? How is it inoculated? How are the tests performed? What is the purpose of each ingredient? What are the possible reactions that could occur? What does each reaction mean?
The Methyl Red (MR) test applied in combination with the Voges-Proskauer (VP) test is used to identify bacteria on the basis of their end products formed from metabolism of glucose. Most bacteria metabolize glucose to pyruvate, which is subsequently broken down to a variety of acids (mixed acid pathway) or butanediol (butylene glycol pathway).
VP test
The VP test detects the fermentation intermediate acetoin a neutral compound. MR and VP are tested from the same medium. The tests are commonly used to differentiate Enterics but can be used to separate bacteria from other groups as well. A red color will indicate the presence of acetoin due to its oxidation to a diacetyl compound. If acetoin has not been produced it will stay yellow.
Know the advantages of Petrifilm.
The advantages are its convenience, ease of use, small size, and stability
Know the limitations of Petrifilm.
The disadvantages are its cost, its restriction to a 1 ml sample volume, and the lack of significant colony morphology.
lag phase
The initial phase, refers to the fact that cell division does not occur immediately, the microorganisms must adjust to their new medium. The length of the stage depends on the conditions of the cells at the time of inoculation and the type of medium that are inoculated into.
Understand the purpose for how to correctly interpret the oxidase test. How is it performed? What are the possible reactions that could occur? What does each reaction mean?
The presence of a c-type-cytochrome correlates with a positive oxidase test. This test can be used to separate the enterics (gram negative that lack cytochrome c oxidase) from gram negative non-enterics. The oxidase agent (tetra-methyl-paraphenylene diamine HCl) acts as an artificial electron donor for the cytochrome c oxidase enzyme, turning a pink color when oxidized. Place 1 drop is oxidase reagent on an isolated colony, oxidase positive colonies typically exhibit a color change from pink to maroon to black. Oxidase negative colonies do not change color.
Understand how to set up dilutions for bacterial quantification
Transfer culture into a dilution bank. Aliquots of the dilutions are plated onto plate count agar with sterile pipettes and spread with hockey sticks, incubated at 37 degrees C and number of colonies counted. It is important to remember that the various dilutions prepared for a viable plate count all derived from a single original suspension of bacteria.
Know the purpose for each type of media used in MB303.
Working with 6 different bacteria, plating them on different types of media and under different environmental conditions, to determine their preferences.
Sodium lauryl sulfate
a detergent that inhibits the growth of gram positive bacteria
Tryptone:
a pancreatic digest of casein. It contains 12% total nitrogen and 5% NaCl.
Tryptose
a peptone derived from meat protein. It contains 12% total nitrogen and 6% NaCl.
Mannitol:
a sugar alcohol, fermentable by some microbes to yield acid by-products
Aerotolerant anaerobe:
an anaerobe which does not utilize oxygen as a final electron acceptor but can grow and tolerate the presence of oxygen. This organism can grow everywhere in a motility tube, with equal amounts of growth throughout.
Strict or obligate anaerobe:
an anaerobe which is killed by the presence of oxygen. This type of anaerobe will only grow below the penetration point of the oxygen, typically in the bottom of the tube.
Beef extract:
an extract of meat used as a nutrient in media, containing amino acids, low molecular weight peptides, nucleotides, organic acids, vitamins, minerals and trace elements. It also contains water soluble proteins and glycogen. This provides energy, all essential macro and micronutrients and growth factors for chemoorganoheterotrophs.
Yeast extract:
an extract of yeast rich in B vitamins and amino acids. It contains numerous unidentified components. Typically its used to provide growth factors in a medium.
Brom Cresol Purple:
an indicator of pH range 5.2-6.8. It is yellow at an acidic pH and purple at a neutral and basic pH.
Brom Thymol Blue
an indicator of pH range 6.0-7.6 yellow green blue. It is green at neutral pH, yellow at an acidic pH and blue at a basic pH. Typically inhibits the growth of gram positive bacteria.
Phenol red
an indicator of pH range 6.6-8.4. It is pink at a neutral pH, yellow at an acidic pH, and hot pink at a basic pH.
Peptone
an ingredient used in many complex media, a hydrolyzed protein that contains many amino acids and minerals. The hydrolyzed protein is prepared from partial proteolytic digestion of meat, casein, soyameal, gelatin or other protein source. Serves as sources of energy, C, N, C and some micronutrients and amino acid growth factors for chemoorganoheterotrophs.
Citrate:
an intermediate of the TCA cycle, can be used as a carbon source by some microbes
Microaerophiles:
an organism that cannot grow in the normal 20% oxygen concentration found in the atmosphere but instead grows in a limited oxygen environment of 2-10%. This organism will form a thin band of growth in a motility tube, a short distance below the agar surface.
Aerobe:
an organism that grows in the presence of oxygen and is able to use oxygen as a final electron acceptor in respiration. An aerobe will only be able to grow on the very surface of motility agar with no growth down in the tube at all
Facultative anaerobe:
an organism that prefers to grow in the presence of oxygen utilizing oxygen as the final electron acceptor, but in the absence of oxygen, it can ferment or undergo anaerobic respiration. This organism can grow everywhere in a motility tube but there should be increased amounts of growth where oxygen is present.
CHONPS
are needed in macro quantities
K, Mg, Ca, Na, and Fe
are needed in micro quantities and a number of additional elements are needed in trace amounts.
Temperature impact
as temperature increases, this will increase enzymatic activity, which will result in an increase in growth rate. However, if the temperature gets too hot, it could inhibit growth rates because it will start to denature the bacteria. A temperature drop might result in a lag phase, as the bacteria have to adjust to the new, colder temperature. Physiologically, during a lag phase, bacteria might experience inhibition of DNA, RNA and protein synthesis.
Citrate tube:
citrate utilization tested using a citrate slant, indicates that the organism makes a citrate permease enzyme (citrase) that allows it to use citrate as a sole source of carbon and energy. The slant will change from green to blue due to the presence of the indicator bromthymol blue, which turns blue at a basic pH. Blue = positive for citrase.
Defined:
composed of designated amounts of specific compounds, the exact contents are known, down to individual elements.
Complex:
contain nutrient rich substances such as yeast extract, peptone, or tryptone and therefore the precise chemical constituents are unknown and will vary slightly from batch to batch
Selective:
contains particular ingredients (salts, dyes, etc) that are used to prevent the growth of specific microbes or it simply lacks ingredients that most microbes require.
Agar:
dried mucilaginous substance extracted from seaweed and used to solidify media. Because of its complex structure, it is difficult for most bacteria to degrade. It melts at 90-100 degrees C and after melting will not re-solidify until 45 degrees.
stationary phase
growth ceases. Normally this occurs once the population level has reached 10^9 cells/ml. This stage is reached because available nutrients are depleted, toxic waste products have accumulated, physical space is limited and or/quorum sensing has occurred.
Absorbance
how much light is absorbed by the sample or in case of bacteria how much light is scattered
Transmittance -
how much light passes through the sample
death or decline phase
if incubation continues , whereby the number of viable cells decreases exponentially.
Tryptophan broth:
indole is produced from the breakdown of tryptophan by the enzyme tryptophanase. Kovac's reagent added, A cherry red ring on the top layer of the tube is indicative of the presence of indole. If indole is not produced a yellow ring will form on the top layer of the tube, indicating that the organism does not make tryptophanase.
All purpose:
meaning it contains all the general ingredients required for growth of non-fastidious microbes and it doesn't specifically exclude growth in any way.
Non-differential:
media that lacks any specialized ingredients to distinguish different microbes.
exponential phase
microorganisms are growing and dividing at the maximal rate possible given their genetic potential, the nature of the medium and environmental conditions. The population is most uniform in terms of chemical and physiological properties. During this stage the generation time (doubling time) can be determined directly from a graph of log cell number versus time.
Nutrient level impact
nutrients are necessary for microbial growth. The results showed that E. coli had better growth rates in the BHI broth as opposed to the minimal salts broths. The salt broth appears to be an unfavorable environment as growth was inhibited. Physiologically, cells become larger during periods of rapid growth. There is an increase in DNA, RNA and protein synthesis.
autotrophs
obtain their carbon from an inorganic source,
Chemolithoautotrophs
obtain their energy from inorganic compounds such as H2, NH3, H2S and use CO2 as a carbon source.
Proteose peptone
one of the kinds of peptone
Lactose broth:
some gram positive bacteria can utilize the disaccharide lactose, particularly any belonging to the lactic acid bacteria group. The ability to ferment lactose will be tested by looking for acid production (a fermentation end product) in tubes of lactose broth that contains only lactose as a carbon and energy source. Bromcresol purple is the indicator. It is purple at a neutral pH and yellow at acid pH so the tubes will start as a purple color and become yellow if lactose fermentation has occurred.
Casein:
the principle protein of cow's milk.
If the organisms only grows on the surface (motility)
the test is inconclusive
Urea broth:
urea is a waste product of protein metabolism in many animals and is excreted in the urine. Some bacteria have an enzyme, urease, which breaks down urea into ammonia and carbon dioxide. The pH of uninoculated urea is 6.8. During incubation, bacteria that make urease will breakdown the urea producing ammonia, which causes the pH to rise. At an alkaline pH the urea broth will change from light orange to dark pink. If the tube is pink urease enzyme is produced. If tube is yellow or orange it is not produced.
Basic requirements for growth:
water; source of energy; source of carbon, nitrogen and other essential elements; required growth factors (organic compounds such as amino acids or vitamins that the microbe cannot synthesize on its own); and the correct physical envionrment
Antibiotic chloramphenicol impact
when comparing treatment 1 and treatment 4 with the chloramphenicol inhibitor, it is easy to see a significant decrease in the colony count for the E. coli that was exposed to the inhibitor. Chloramphenicol works by inhibiting protein synthesis. Physiologically, it prevents chain elongation by inhibiting the peptidyl transferase activity of the bacterial ribosome.
Differential:
where the appearance of the microbes on the media will vary depending upon their interaction with specific ingredients in the media. The difference can either be in colony color or a change in the medium itself, such as a color change or clearing. This is usually achieved with pH indicators that change color with different pHs.
motile
will move away from the stab line. Will make the entire medium turbid. If you are unsure if the entire tube is turbid or not, compare it to a tube or uninoculated motility agar.
non-motile
will only grow in the stab line. The stab will appear 3D when rotated, often the stab has a dagger like appearance where its thicker in one view and then very thin in another view. This is due to the fact that the needle often moves slightly from side to side during inoculation
