Quiz 2: Microbiology Lab

Chemical Agents of Control: Chemotherapeutic Agents

*To determine a therapeutic drug of choice, determine its mode of action, possible adverse side effects in the host, & the scope of its antimicrobial activity. - Mode of action: interfere w/ microbial metabolism (producing a bacteriostatic or bactericidal effect on the microbes w/o producing a like effect in the host). - Chemotherapeutic agents act on multiple cellular targets; mechanism of action: inhibition of cell wall synthesis, inhibition of nucleic acids, inhibition of folic acid synthesis, & disruption of cell membrane. - Drugs can be separated into 2 categories: 1. Antibiotics: synthesized and secreted by some true bacteria, actinomycetes, & fungi that destroy/inhibit growth of other microorganisms; some are laboratory synthesized/modified ~ origins are living cells. 2. Synthetic Drugs: synthesized in the laboratory.

Physical Factors: Temperature

- Effect of temp. on bacterial growth & gene expression - Increasing temperatures cause enzymes denaturation of their protein structure; decreasing temperatures cause enzyme inactivation (cellular metabolism diminishes). - Cardinal (significant) temperature points: the maximum, minimum and optimum temperature range within which the seed of a particular species germinate (reproduce). - Minimum Growth Temperature: lowest temp. needed for growth to occur. - Maximum Growth Temperature: highest temp. needed for growth to occur. - Optimum Growth Temperature: the temp. where the rate of reproduction is most rapid; cells dividing very quickly; population expansion at its max. - All bacteria classified into 1 of these major groups: 1. Psychrophiles: grow at 20 C or below (0 C - 20 C) 2. Mesophiles: grow betw. 21 C - 45 C; human body temperature (37 C) - Psychrotrophs: mesophiles w/ slightly lower temp. ranges; 20 C - 30 C. 3. Thermophiles: grows above 45 C - The ideal temp. for specific enzymatic activities may not coincide w/ the optimum growth temp. for a given organism

Mode of Action vs. Mechanism of Action

A mode of action (MoA) describes a functional or anatomical change, resulting from the exposure of a living organism to a substance. In comparison, a mechanism of action (MOA) describes such changes at the molecular level. Mode of action of a biomolecule entering the body refers to the mode in which the action is brought about and is characterized by the changes that take place in terms of physiological aspects. Mechanism of action refers to the process by which a substance undergoes bio chemical changes within the host in order to bring about the specific action of the administered substance.

Kirby-Bauer Method

- A standardized diffusion procedure with filter-paper discs on agar; used to determine the drug susceptibility of microorganisms isolated from infectious processes. - Zone of inhibition/Zone of clearing's diameter is measured to determine how effective the antibiotic is on the microorganism. - Susceptibility of an organism to a drug is assessed by the size of this zone, which is affected by other variables: 1. ability & rate of diffusion of the antibiotic into the medium & its interaction w/ the test organism. 2. number of organisms inoculated 3. growth rate of the organism *Test organism is resistant, intermediate, or susceptible to the antibiotic. - Filter-paper discs are infused w/ dif. antibiotics & then placed on the surface of a Mueller-Hinton agar plate that has been inoculated w/ bacteria by means of a cotton swab; an antibiotic disk dispenser is used to aseptically apply the antibiotics to the agar plate at well-spaced intervals.

Oxygen Conditions

- Aerobic: Requires oxygen. - Candle Jar: Burns some oxygen; reduces oxygen, produces more CO2. - Anaerobic: No oxygen required.

Isolation of Fungi from Soil Experiment

- Before a fungal specimen may be characterized or described, it must be isolated from other bacteria or fungal species that are present. 1. Transfer 1g of soil to one of the 50mL tubes & add 45mL of sterile water. 2. Vortex the sample to evenly suspend soil particles in water for about 3 minutes. 3. Allow the soil suspension to sit for 15 minutes so the debris and large soil particles settle out. 4. While the solution is settling, label the PDA plates as #1 through #4 & add 50mL sterile water to the second tube. 5. Using a sterile Pasteur pipette, transfer 1 mL of soil suspension to plate #1 & 100 microliters to plate #2. 6. Transfer 1 mL soil suspension to the second tube with 50 mL & vortex for 1 minute to mix. 7. From the second tube, transfer 1 mL of suspension to plate #3 & 100 microliters to plate #4. 8. Using a sterile L-spreader, start w/ plate #4 and spread the soil suspension evenly across the plate surface, then continue to plate #3, then #2, then #1. 9. Seal plates with Parafilm & incubate at room temperature for 7-10 days. Agar side down

Chemical Agents for the Control of Microbial Growth

- Control of microbial growth is essential to prevent & treat disease and to inhibit the spoilage of foods & other industrial products; chemical & physical agents adversely affect microbial structures and functions producing a microbicidal (kills the microbes immediately) or microbistatic (inhibits the reproductive capacities of the cells & maintains the microbial population at a constant size) effect. - Chemical Methods: 1. Antiseptics: used on living tissue that kill or inhibit the growth of vegetative (reproduction or propagation achieved by asexual means) microbial forms. 2. Disinfectants: kill or inhibit the growth of vegetative microbial forms on nonliving materials. 3. Chemotherapeutic Agents: destroy or inhibit the growth of microorganisms in living tissues; chemical substances used to treat infectious diseases.

Methods to Isolate Fungal Growth

- Direct Transfer/Direct Plating: taking a small sample from a visible growth that is relatively ubiquitous (found everywhere) & transferring it directly to an agar plate for growth in a lab; allow for growth for all organisms present but may not allow for individual species to be directly isolated. - Dilution Plating: may allow isolated mycelium to grow & be transferred for pure cultures (for samples that may be more complex in the potential numbers of species present).

Experiment: TSA Plates - Temperature & Oxygen Requirements

- If the bacteria was supposed to produce a pigment to obtain a color but it remained colorless then there was contamination involved with the bacteria. - Bacteria could have also been transferred from other cultures, which can alter the results. Temperature: - 4 TSA plates - bacteria: Ec, Gf, Sm, Pf - 4 C, 21 C, 37 C, 55 C Results: - Ec: grows in 21 C & 37 C - Sm: grows in 21 C & 37 C - Gf: grows in 55 C - Pf: grows in 4 C & 21 C Oxygen: - 3 TSA plates - bacteria: Ec, Cs, Ml, Ll - aerobic, candle jar, and anaerobic Results: - Ec: facultative anaerobe (grew in all environments). - Cs: obligate anaerobe (only grew in anaerobic conditions). - Ml: obligate aerobe (only grew in oxygen & candle jar conditions). - Ll: facultative anaerobe (grew in all environments). Label the agar plates and divide it into quadrants (on the bottom of the agar plate). Used the aseptic technique to transfer the bacteria onto the agar plates; the agar plate is streaked with the bacteria. Lastly, incubate the bacteria in their specific requirements for temperature and oxygen.

Kombucha (S.C.O.B.Y) Experiment

- Jar Ingredients: - 400mL of boiled H2O - 3 spoonfuls of sugar - 1 bag of black tea - add 300ml of cool H2O - add 50ml of Kombucha - paper towel & rubberband (if you don't have a cover/lid for the jar) - S.C.O.B.Y. (Symbiotic Culture of Bacteria and Yeast) - A SCOBY is a cellulose-based biofilm that results in the natural fermenting process of making kombucha. It forms together when you ferment the lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeast together. - Works wonders in the brewing of kombucha. SCOBY is made up of a large collection of different bacteria and yeast species, which live together in a symbiotic relationship on a large layer of cellulose - a cellulose mat that houses the bacteria and yeast cultures that turn sweet tea into kombucha

Antibiotics Experiment (Bacterial Lawn)

- On the 3 Mueller-Hinton Agar plates, you will create a bacterial lawn (no single colonies) of Ec, Pa, and Sa bacteria; a cotton swab is used to aseptically transfer the bacteria to the medium (press the cotton swabs against the wall of the test tubes to remove excess liquid) ~ work near flame. Flick the bacteria before obtaining them with the cotton swabs. - Using the antibiotic disk dispenser aseptically apply the antibiotics to the agar plate; use sterile tweezers/forceps to gently tap the antibiotic discs to solidify their placements in the agar. - The bottom of the agar plate should be facing up; the lid/cover of the plate is at the top (facing up) instead of the bottom (inverted position).

Antibiotics: Penicillin G (P10), Cephalothin (KF30), Erythromycin (E15), Neomycin (N30), Bacitracin (B10)

- Penicillin G: weakens peptidoglycan layer (cell wall); Ec, Sa, & Pa are all resistant. - Cephalothin: inactivates penicillin-binding proteins (PBP) located on the inner membrane of the bacterial cell wall; Ec & Pa are resistant, Sa is Intermediate. - Erythromycin: inhibition of protein synthesis; Ec & Pa are resistant, Sa is susceptible. - Neomycin: inhibits bacterial protein synthesis; Pa is resistant, Ec & Sa are susceptible. - Bacitracin: inhibits bacterial cell wall synthesis. Zone Diameter Interpretive Standards: measure one side of the clearing to the other side. Penicillin G: - Resistant: staphylococci: <=28; other bacteria: <=14 - Intermediate: staphylococci & other bacteria: N/A - Susceptible: staphylococci: >=29; other bacteria: >=15 Cephalothin: - Resistant: <=14 - Intermediate: 16-17 - Susceptible: >=18 Erythromycin: - Resistant: <=13 - Intermediate: 14-22 - Susceptible: >=23 Neomycin: - Resistant: <=12 - Intermediate: 13-16 - Susceptible: >=17 Bacitracin: - Resistant: <=8 - Intermediate: 9-12 - Susceptible: >=13

Physical Agents for the Control of Microbial Growth

- Physical Methods: modes of action of the dif. chemical & physical agents of control vary; all produce damaging effects to 1 or more essential cellular structures/molecules (cause cell death/inhibition of growth); sites of damage that can result in malfunction are: cell wall, cell membrane, cytoplasm, enzymes, & nucleic acids. Harmful effects are shown within the following ways: 1. Cell-Wall Injury: can occur in 1 of 2 ways; first, lysis (breakdown) of the cell wall will leave the wall-less cell - this is called a protoplast (susceptible to osmotic damage & a hypotonic environment cause lysis of the protoplast). Second, certain agents inhibit cell wall synthesis (results in an unprotected protoplast). 2. Cell-Membrane Damage: lysis of the membrane (cause immediate cell death); selective nature of the membrane is affected (loss of essential cellular molecules or interference w/ the uptake of nutrients). 3. Alteration of the Colloidal State of Cytoplasm: Certain agents cause denaturing of cytoplasmic proteins. 4. Inactivation of Cellular Enzymes: inactivated competitively or noncompetitively: - Noncompetitive Inhibition: irreversible & occurs after some physical agent is used (results in the uncoiling of the protein molecule ---> biologically inactive). - Competitive Inhibition: reversible & occurs when a natural substrate is forced to compete for the active site on an enzyme surface w/ a chemically similar molecular substrate (blocks the enzyme's ability to create end products). 5. Interference w/ the structure and function of the DNA molecule: DNA molecule is the control center of the cell and represents a cellular target area for destruction or inhibition; some agents have an affinity (liking) for DNA (cause breakage or distortion of the molecule interfering w/ its replication & protein synthesis).

Antiseptics Experiment

- There are 3 TSA plates for Ec, Pa, and Sa; on these plates create 4 quadrants on the bottom of the agar. - There are various antiseptics you can choose from, pick 4 of them to put on all 3 TSA plates; there are blank discs provided that you will soak into the antiseptics of your choosing using sterile tweezers and place onto the agar plates (gently tap each disc with the tweezers). - The bottom of the agar plate should be facing up; the lid/cover of the plate is at the top (facing up) instead of the bottom (inverted position).

Shake-Tube Inoculation

- the oxygen needs of microorganisms can be determined by noting their growth distributions in this procedure - this procedure requires the inoculum to be placed in a melted agar medium; next, shaking of the test tube (rapidly rotating) to disperse microorganisms throughout the agar; then, rapid solidification (ice waterbath) of the medium to ensure the cells remain dispersed; lasty, incubate at 37 C. - aerobes exhibit surface growth. - anaerobes exhibit growth in the bottom of the deep tube. - facultative anaerobes exhibit growth throughout the medium; when oxygen is present growth is more abundant and closer to the surface of the tube; when oxygen is absent growth is closer to the bottom of the tube. - microaerophiles exhibit growth slightly below the surface. - aerotolerant anaerobes exhibit growth uniformly/equally throughout the medium.

Physical Factors: Atmospheric Oxygen Requirements

5 major groups: 1. Aerobes: requires oxygen for growth; oxygen is used as the final hydrogen (electron) acceptor. 2. Microaerophiles: requires limited amounts of oxygen for growth; an excess of oxygen blocks the activities of their oxidative enzymes (results in death). 3. Obligate Anaerobes: doesn't require oxygen for growth (other molecules act as the final hydrogen (electron) acceptor); presence of oxygen results in the formation of toxic metabolic end products (i.e., radicals); don't produce the enzymes: superoxide dismutase & catalase. 4. Aerotolerant Anaerobes: fermentative organisms that don't use oxygen as a final electron acceptor; produce the enzymes: superoxide dismutase and/or catalase (not killed by the presence of oxygen). 5. Facultative Anaerobes: can grow in the presence or absence of oxygen (they grow better in oxygen rich environments); oxygen present - aerobic respiration, oxygen absent - cellular respiration.

Disinfectants & Antiseptics

The efficiency of all disinfectants & antiseptics is influenced by a variety of factors: 1. Concentration: higher concentrations producing a more rapid death. Cannot be arbitrarily (randomly) determined; the toxicity of the chemical to the tissues being treated & the damaging effect on nonliving materials must also be considered. 2. Length of Exposure: all microbes are not destroyed within the same exposure time; the longer the exposure to the agent, the greater its antimicrobial activity. Sensitive forms are destroyed more rapidly than resistant ones. Toxicity & environmental conditions of the chemical must also be considered in determining the length of time necessary. 3. Type of Microbial Population to be Destroyed: bacterial spores - most resistant forms; capsulated bacteria - more resistant than noncapsulated forms; acid-fast bacteria - more resistant than non-acid-fast; older, metabolically less-active cells - more resistant than younger cells. - Type of microorganisms present influences the choice of agent. 4. Environmental Conditions: a. Temperature: chemical reaction between the agent & cellular components ---> cells are killed; increasing temp., increases the rate microbial populations are destroyed. b. pH: may affect the microorganisms and the compound. Extremes in pH are harmful ---> may enhance the antimicrobial action of a chemical; deviation from a neutral pH may cause ionization of the disinfectant, (depending on the chemical agent) may increase or decrease the chemical's microbicidal action. c. Type of Material on which the Microorganisms Exists: destructive power of the compound on cells is due to its combination w/ organic cellular molecules. If the material on which microorganisms are found primarily organic (i.e., blood, pus, tissue fluids) the agent will combine w/ these extracellular organic molecules & the agent's antimicrobial activity will be reduced.

Oxidation-Reduction (Redox) Reaction

a type of chemical reaction that involves a transfer of electrons between two substances.

Reduction

gaining a hydrogen & its electron

Oxidized

substance loses a hydrogen ion & its electron

Respiration

the oxidation of substrates for energy necessary to life