Practice problems Exam #1

You inoculate a culture of Bacillus cereus with exactly 100 bacterial cells. After 3 hours, your sample contains 6,400 bacterial cells. How many generations have occurred? What is the generation time for this population?

#Generations = log #cells(end) -log#cells(start) _____________________________ 0.301 (=log2) Generation Time= 60min/hr x #hrs __________________ #generations

Describe the four actions by which microbial control agents affect microorganisms. How do chemical germicides work to control microorganisms? Give one example of a physical or chemical treatment that can control microorganisms by one of each action. (You should be familiar with the action by which each treatment controls microorganisms).

*Sterilants -Destroy all microbes Ex. Autoclave (physical) -Denatures protein. *High-level disinfectant -Destroy all microbes except endospores. Ex. Boiling (physical) Denatures protein. *Intermediate-level disinfectant -Destroy all vegetative bacteria, fungi & most viruses. Ex. Alcohol (chemical) -Denatures protein, damages membranes. *Low level disinfectants. -Destroy fungi, enveloped viruses, and all vegetative bacteria except from mycobacteria. Ex. Ammonium compounds (chemical) -soap/detergents, mechanical removal of microbes from surfaces. Chemical germicides damage bacteria trough: - Damage to nucleic acid - Protein denature/loss of function - Alteration of cell wall - Alteration of cell membrane permeability

Name three classes of antimicrobial medications that target protein and describe how each works.

-Aminoglycosides -Cause misreading of mRNA by blocking the initiation of translation. -Tetracyclines -Block the attachment of tRNA to the ribosome -Macrolides -Prevent the continuation of protein synthesis

Draw and label the four main phases of growth in a bacterial growth curve. Describe the key events occurring within the culture as it passes through each stage and which phase can be used to determine generation or doubling time.

1 Lag phase -Preparation for growth -Not actively dividing 2 Log phase -logarithmic/exponential growth -Most susceptible to antibiotics that inhibit cell wall synthesis. -Can determine generation time/doubling time. 3 Stationary phase -#new cells = # deaths 4 Death/Decline phase -Accumulation of waste, loss of nutrients overpopulation... #deaths >new cells.

Describe the four general mechanisms of antimicrobial resistance. Are there any similarities to any of the four actions by which microbial control agents affect microorganisms (Ch. 5)?

1. increased elimination of antibiotic (efflux pump ejects antibiotic) 2. Decreased uptake of antibiotics-porin proteins prevent antibiotic entry into the cell 3. alter target molecule (shape)-antibiotic can no longer bind 4. antibiotic inactivating enzyme (β-lactamase)

Draw the chemical composition of the cell wall of a gram-positive (G+) and gram-negative (G-) bacterium. a. Label each component. b. Briefly describe how each cell wall is different from the other. c. Which cell type is more susceptible to antibiotics such as penicillin (G+ or G-)? Explain why.

A. On test: label and recognize something like p. 3 in handout B. Gram Positive (G+) cell wall (stains purple) -Thick peptidoglycan layer -Peptide interbridge -Teichoic acids -Suceptible to penicillin & lysosome Gram Negative (G-) cell walls (stains pink) -Thin peptidoglycan layer -Outer membrane: *Barrier to antibiotics or other, consist of: -LPS (Lipopolysaccharide) layer: -O antigen -Core Polysaccharide -Lipid A (endotoxin) Peptidoglycan layer= Peptide bridge(of amino acids) + Glycan (modified sugars NAM-NAG repeats. C. Gram positive -because it doesn't have the outer membrane, which works as a protective barrier, penicillin wants to go after the peptidoglycan layer.

Which cell type (G+ or G-) are β-lactam antibiotics most effective against? Explain why. How do β-lactam antibiotics work on these cells? Why is this class of antibiotics only bactericidal to growing bacteria? Considering that all β-lactam antibiotics have the same target, why do they vary in their spectrum of activity? What is MRSA and why is it significant?

B-lactam antibiotics target the peptidoglycan layer and inhibit enzymes to help form links between glycan chains. G+ cells don't have an outer membrane so the peptidoglycan layer is exposed and therefore more sensitive than G- bacteria that have some protection from its outer membrane. -B-lactam antibiotics are only bactericidal to growing bacteria because they continuously synthesize peptidoglycan. -They vary in their spectrum of activity because of resistance, if one factor inhibits the antibiotic from functioning properly then another one in the spectrum might work better. -MRSA =methicillin-resistant Staphylococcus aureus. It is significant because it is resistant to methicillin as well as nearly all other β lactam antibiotics.

Clostridium and Streptococcus are both catalase-negative. Streptococcus grows by fermentation. Why is Clostridium killed by oxygen whereas Streptococcus is not? Explain in terms of the oxygen requirements for each organism (categorize each organism based on their oxygen requirement), the pattern of growth for each, as well as key players in the growth of each.

Clostridium is an obligate anaerobe, which means that it cannot multiply if O2 is present. Obligate anaerobes harvest energy using processes other than aerobic respiration. Streptococcus are aerotolerant anaerobes and are indifferent to O2. They can grow in its presence, but do not use it to harvest energy. They are also called obligate fermenters, because fermentation is their only metabolic option.

Explain why it takes longer to kill a population of 109 cells than it does to kill a population of 103 cells. Please define any terms described here and their relevance to your response.

D value is the time required for killing 90% of a bacterial population under specific conditions. One D value reduces the number of cells by one order of magnitude. Since 109, is a bigger about of cells that 103 it also requires more time. D value = Decimal reduction time

Under what circumstance would the use of filtration be used instead of autoclaving? Describe each process (filtration and autoclaving). Which sterilization process (give specific name) would kill both vegetative cells and endospores? Does filtration remove all microorganisms? Please explain your response.

Filtration is good for air and heat sensitive liquids. Filtration -Membrane filters physically remove microbes. Vacuum pull liquids trough filter and the microbes stays in the filter. Autoclaving -Hig pressure in a chamber allow for high heat. Steam reach 121C and sterilize by denature proteins in 15 minutes. Autoclaving would kill both endospores & vegetative cells, filtration do not kill, only eliminate. Standard filtration do not remove mycoplasma, that require smaller pore size. When pore size is small enough to remove all kind of microbes the liquid will be sterile.

Which type of microscopy technique might be useful for the rapid detection of a specific organism? List the name and describe the principles behind how such a technique is performed to aid in the detection of an organism.

Fluorescence Microscopy -a light microscope that detect fluorescence. Some organisms have natural fluorescence, others can be tagged with a fluorescence marker: An fluorescence marker is attached to the antibody which have a unique shape that bind to a specific bacteria. When they bind the fluorescence dye molecule stand out/"light up" under the UV-light. Fluorescence that cant bind won't show. That way we can detect specific bacteria. For example: Want to find E. coli? Add antibody's that specifically bind to e-coli, if their fluorescence marker "light up" E. coli is present.

Why are Gram-positive bacteria usually less resistant to chemical biocides than Gram-negative bacteria? Why are bacterial endospores usually among the most resistant? Which treatment(s), physical and/or chemical, would be used to destroy Gram-positive cells, Gram-negative cells, and bacterial endospores? Please provide rationale for your response.

Gram positive bacteria do not have a protective membrane wich makes them more sensitive than Gram negative bacteria. Endospores have many layers of protection and can therefore resist tough environments that would kill other spieces. -Sterilants kill all 3, such as Autoclave & Dry heat. Filtration can sterilize by elimination but do not kill/destroy.

You are working in a clinical laboratory and you need to examine an unstained urine sample for the presence of bacteria. Briefly describe the type(s) of light microscope(s) that could be used to observe this specimen and how they function.

It would be best to use one of the light microscopes that increase contrast; they require no staining so specimen is alive. Dark field -Dark background light specimen. Phase contrast -Beams of light at an angle, cells and dense material appears darker. It shows a clear, sharper image than dark field. DIC (Differential interference contrast) -Separates beams of light into 2, hit specimen, then 2 recombine. This creates a 3D-effect with areas of color.

Microorganisms require phosphorus, sulfur, iron, and magnesium for metabolism. What specifically are each of these elements used for in metabolism?

Phosphorous -Found in nucleic acids, membrane lipids, and ATP. Sulfur-Component of amino acids Magnesium -For enzyme function. Iron -Part of certain enzymes.

Describe the three basic components and function of a prokaryotic flagellum. What does the term chemotaxis mean? How does a flagellum move to achieve chemotaxis in response to a nutrient and a toxin (describe in terms of "runs" and "tumbles")?

Prokaryotic flagellum -The flagella is for motility. Many pathogens have flagella for movement. It has 3 components: *Basal body: Rotor powered by proton-motive force (H+ Movement), extends from cytoplasm to outer membrane. *Hook -gives the flagellum flexibility to rotate. *Filament -Made of flagellin subunits (Protein) Chemotaxis = Movement in response to chemical. Chemoattractant (i.e. nutrient) -moves towards (more runs than tumbles) Chemorepellent (Toxin) -Away/confused movement. (more tumbles than runs) Tumble=clockwise rotation Run=counterclockwise rotation