Microbio Lab Exercise 24: Chemical methods of Control: Disinfectants and Antiseptics

Antimicrobial agents

(chemicals that control the growth of microbes) wide varieties of chemicals that are available for controlling growth of microbes.

what is macrolides? -products of actinomycetes or semi-synthetic derivatives

-used on those allergic to penicillin

Broth dilution: What is the minimum bacteriostatic concentration?

A = 1:190 B = 1:160

Broth dilution: What is the minimum bactericidal concentration of each antibiotic?

A = 1:80 B = 1:150

What is the decolorizing agent in the acid-fast stain?

Acid-alcohol

Enterotube Chart

Add all the circled numbers in each bracketed section and enter the sum in the space provided below the arrow on your Results page. -Locate the 5 digit number in the Computer Coding and Identification System booklet -Find the best identification in the column entitled "ID Value."

The Enterotube (used to identify Enterobacteriaceae):

All "fake urines" are members of the Family Enterobacteriaceae. 1) Escherichia coli (E. coli) 2) Enterobacter aerogenes 3) Proteus mirabilis 4) Citrobacter freundii 5) Klebsiella pneumonia 6) Enterobacter cloacae

What is the standard method for measuring the effectiveness of a chemical agent called

American Official Analytical Chemist's use-dilution test

Which antibiotic is more effective against Staphylococcus aureus?

B

What are the Gram reactions of Bacillus?

Bacillus = gram-positive

Those agents that result in bacterial death are called what

Bactericidal agents

If most of the normal microbiota and transient microbiota aren't harmful, then why must hands be scrubbed before surgery?

Because externally they may not be harmful but internally they can cause infection

In the disk-diffusion technique measuring bacteriostatic or bactericidal activity?

By definition, antibiotics are growth-inhibitory, but some are bactericidal. To be sure, subculturing would be required, after removal of the antibiotic.

The use-dilution test cannot be used to evaluate what compounds? Which compounds can they evaluate?

CANT--> Bacteriostatic Compounds CAN--> Bactericidal Compounds

How might the acid-fast characteristic of Mycobacterium enhance the organism's ability to cause disease?

Cells are not digested by phagocytes Cell walls contain lipids (mycolic acid) that resist drying and environmental stress so bacteria can survive harsh conditions

Disinfectants

Chemical agents used on inanimate objects to lower the level of microbes on their surfaces

List the steps of the Gram-stain procedure in order (omit washings), and fill in the color of gram-positive cells and gram-negative cells

Chemical gram-positive/ gram-negative 1. Crystal violet purple/ purple 2. iodine (mordant) purple/purple 3. alcohol (ethanol) violet/ clear 4. safranin violet/ red

In the use-dilution test, a chemical is evaluated by its ability to kill 10 power of 6 dried Clostridium sporogenes or Bacillus subtilis endospores. Why is it considered a stringent test?

Chemicals do not readily penetrate bacterial endospores, and the number of endospores used is higher than would be expected in normal use of disinfectant.

How could the procedures used in this experiment be altered to measure bacteriostatic effects?

Could use a subculture instead, or a diluted substance w/o the test chemical.

How would you distinguish between a bacteriocidal & bacteriostatic microbe?

Culture it, have a control, test if its bacterocidal or bacterostatic w/ a test substance, culture it again. If there is no growth on the SECOND subculture it is bacteriocidal & if there is growth it is Bacteriostatic

Before the disinfectant is selected, the ...... for the most common and persistent microbes identified at a healthcare facility should be determined

Decimal reduction time (DRT)

Mueller-Hinton agar allows the antimicrobial agent to do what?

Diffuse freely

What is phenol (carbolic acid), and what is its usual application

Disinfectant 5%

What is another name for Kirby-Bauer test?

Disk diffusion test

Thermal Death Time (TDT)

Divide nutrient agar plate into 5 sections. Using aseptic techniques inoculate 0 section place in water bath after 30 sec. Inoculate 30 sec. section, after 1-1/2min inoculate 2 min. section. After 3 min inoculate 5 min. section. After 10 min. inoculate 15 minutes. What test procedure is this?

What happens during incubation of the Petri plates in the Disk-diffusion menthod

During incubation, the antimicrobial agent diffuses from the disk, from an area of high concentration to an area of lower concentration. An effective agent will inhibit bacterial growth, and measurements can be made of the size of the zones of inhibition around the disks.

What is the decolorizing agent in the Gram stain?

Ethanol

Nonionizing Radiation

Forms of radiant energy such as radio waves, microwaves, infrared light, and ordinary light that do not have enough energy to cause ionization of atoms in living tissue. Compare ionizing radiation.Main absorption wave lengths for green algae, green plants and photosynthetic bacteria.

Antibiotics inhibit growth internally and disinfectants are used for surfaces to kill microbes.

How are antibiotics different from disinfectants?

Incubate the diluted chemical and bacteria overnight. If there is no growth, then the organisms should be subcultured to sterile nutrient medium minus the test chemical. If the chemical is bacteriocidal, no growth will occur. Subculture will grow if chemical was bacteriostatic.

How could the procedures used in the experiment be altered to measure bacteriostatic effects?

SIM

Hydrogen-Sulfide Indole Motility

Enterotube procedure

I. Compartment 1. Interpret the results of glucose fermentation. a. Yellow is positive; red or orange is negative; if positive, circle the number 2 under glucose on your Results page. II. Compartment 1. Interpret the results of gas production. a. Wax lifted from agar is positive; wax not lifted from agar is negative; if positive, circle the number 1 under gas on your Results page. III. Compartment 2. Interpret the results of lysine decarboxylase. a. Any purple is positive; yellow is negative; if positive, circle the number 4 under lysine on your Results page. IV. Compartment 3. Interpret the results of ornithine decarboxylase. a. Any purple is positive; yellow is negative; if positive, circle the number 2 under ornithine on your Results page. V. Compartment 4. Interpret the results of H2S production. a. True black is positive; beige is negative; if positive, circle the number 1 under H2S on your Results page. VI. Compartment 4. Indole production. a. Make a hole in the section of the Enterotube in the section labeled Indol. Place 1-2 drops of Kovac's reagent into the section b. If red- pink color appears on walls or on agar the result is positive; no color change is negative VII. Compartments 5- 8 . Interpret the results of adonitol fermentation, lactose fermentation, arabinose fermentation, and sorbitol fermentation, respectively. a. Any yellow is positive; red or orange is negative; if positive, circle the points granted for each carbohydrate given on your Results page. VIII. Compartment 9. Voges-Proskauer test. a. This test is not used unless a final VP confirming test is later called for IX. Compartment 10. Interpret the results of dulcitol fermentation. a. Yellow is positive; any other color is negative; if positive, circle the number 1 under dulcitol on your Results page. X. Compartment 10. Interpret the results of Phenylalanine deaminase . a. Black or smoky gray is positive; any other color is negative; if positive, circle the number 4 under PA on your Results page. XI. Compartment 11. Interpret the results of urea hydrolysis in a. Red or purple is positive; beige is negative; if positive, circle the number 2 under urea on your Results page. I. Compartment 12. Interpret the results of citrate utilization. a. Any blue is positive; green is negative; if positive, circle the number 1 under citrate on your Results page.

Chemicals do not readily penetrate bacterial endospores, and the number of endospores used is higher than would be expected in normal use of disinfectant.

In the use-dilution test, a chemical is evaluated by its ability to kill 10 power of 6 dried Clostridium sporogenes or Bacillus subtilis endospores. Why is it considered a stringent test?

Log phase

In which growth phase is an organism most sensitive to an antimicrobial agent?

Logarithmic growth phase

In which growth phase is an organism most sensitive to an antimicrobial agent?

What is the cellular target of penicillin?

Inhibits cross-linking of the cell wall's peptidoglycan

What is the cellular target of trimethoprim?

Inhibits purine and pyrimidine synthesis

What is the cellular target of ciprofloxacin?

Interferes with DNA replication

Why is the Kirby-Bauer test valuable?

It is a standard tool for measuring the effectiveness of antimicrobics against pathogenic microorganisms

Is a test chemical or a lab disinfectant more effective?

Lab disinfectant

Disk-diffusion test against bacterium Which drug should be used to treat an infection caused by this bacterium?

Largest zone of inhibition

In which growth phase is an organism most sensitive to an antibiotic? .

Log growth phase. Most antibiotics are effective against actively metabolizing cells

What test did we use looking for staphylococcus?

Mannitol Salt agar (MSA)

To perform a use-dilution test, what happens:

Metal rings are dipped into standardized cultures of the test bacteria grown in liquid media, removed, and dried. The rings are them placed in the disinfectant at the concentration recommended by the manufacturer for 10 minutes at 20 degrees celsius. The rings are then transferred to a nutrient medium to permit the growth of any surviving bacteria.

Suppose you are viewing a Gram-stained field of red rods and purple cocci through the microscope. What do you conclude?

Mixed culture

How might a flagella contribute to pathogenicity?

Motility

The Kirby-Bauer test uses the ______________.

Mueller-Hinton agar

Why is the disk-diffusion technique not a perfect indication of how the drug will perform in vivo? What other factors are considered before using the antimicrobial agent in vivo?

Mueller-hinton medium is not identical to the body. Patient size, age, and other physical conditions. As well as side-effects and other drugs must be considered.

What diseases are diagnosed using the acid-fast procedure?

Mycobacterium

How do normal microbiota and transient microbiota differ?

Normal = permanent microorganisms Transient = present only for days or weeks

What antibiotics did the class test in the Kirby-Bauer test?

Penicillin Chloramphenicol Trimethoprim Ciprofloxacin

What effect would the presence of tetracycline in the body have on penicillin therapy?

Penicillin inhibits cell wall synthesis. Prior use of tetracycline may slow metabolism to the extent that subsequent use of penicillin may not be effective.

What does yellow coloration of the media (MSA) mean?

Presence of coagulase , mannitol being fermented.

What is the cellular target of chloramphenicol?

Prevents peptide bond formation during translation

If you performed a Gram stain on human cells, what would happen?

Primary stain would be removed easily because human cells don't have cell walls

For most purposes, three strains of bacteria are used in the American Official Analytical Chemist's use-dilution test; They are:

Salmonella enterica Choleraesuis, Staphylococcus aeureus, and Pseudomonas aeruginosa

What is a surgeon trying to accomplish with a 10 minute scrub with a brush followed by an antiseptic?

Scrubbing is an attempt to dislodge as many microbes as possible Antiseptic is used to kill as many microbes as possible

What is a surgeon trying to accomplish with a 10 minute scrub with a brush followed by an antiseptic?

Scrubbing is an attempt to dislodge as many microbes as possible Antiseptic is used to kill as many microbes as possible.

Mannitol Salt agar (MSA)

Selective- for Gram (+) and salt tolerant (Staphylococcus epidermidis) stays red color doesn't change. Differential- in that it ferments mannitol and it turns yellow - Staphylococcus aureus

What two different bacteria did we use in the Kirby-Bauer test?

Staphylococcus aureus Escherichia coli

Which organism was most susceptible to most antibiotics?

Staphylococcus aureus Pseudomonas aeruginosa.

Why isn't one antimicrobial agent equally effective against all three bacteria?

Structurally characteristics of cell walls and species specific enzyme may vary.

Mixed culture

Suppose you are viewing a Gram-stained field of red rods and purple cocci through the microscope. What do you conclude?

What effects would the presence of tetracycline in the body have on penicillin therapy?

Tetracycline is bacteriostatic (stops growth) Penicillin needs growing bacteria in order to kill it. So penicillin is useless with tetracycline

The effectiveness of the bacteria can be determined by what after the use-dilution test?

The amount of the resulting growth

Why will gram-positive cells more than 24 hours old stain gram-negative?

The cell wall can't retain primary stain

Suppose you performed a Gram stain on a sample from a pure culture of bacteria and observed a field of red and purple cocci. Adjacent cells were not always the same color. What do you conclude?

The culture is old

The DRT (decimal reduction time) is:

The time it takes to Kill 90% of the test microbial population.

The Kirby-Bauer test for agar diffusion methods minimizes what?

The variance between the laboratories.

You can see endospores by simple staining. Why not use this technique?

They look white or transparent

Considering you can't identify bacteria from a Gram stain, why might a physician perform a Gram stain on a sample before prescribing an antibiotic?

To determine its sensitivity to the antibiotic correlates with cell wall type

Escherichia coli

What bacteria was most acceptable to heat?

The more damage the harder to repair

What is purpose for exposing plate C to 60 sec. of UV light and storing in dark?

It evaluates a chemical under conditions matching it's application. For example, a wall disinfectant would be diluted and used on a contaminated wall. Wet swabs or Rodac plates-nutrient agar pressed against wall would be applied and the resulting growth would be evaluated.

What is the use -dilution method?

Bacillus subtilis- endospores

Which organism had the highest tolerance for UV? What bacteria tolerated the widest range of temperatures? What bacteria was most resistant to heat? Why?

Staphylococcus aureus

Which organism had the lowest tolerance for UV?

E. coli

Which organism is a lactose fermenter?

Serratia marcescens

Which organism is capable of light repair?

Gram (+) organisms

Which organisms tend to be more susceptible to antibiotics in general?

Glucose

Which sugars were used to test for fermentation on SA?

Bacteriostatic

agent cause temporary inhibition of growth.

Bactericidal

agent disinfectants and antiseptics affect bacteria in many ways. Those that result in bacterial death is called an ?

Antiseptics chemical

agent used on living tissue to decrease the number of microbes

Disinfectants chemical

agents used on inanimate objects to lower the level of microbes on their surfaces.

what is tetracyclines's resistance mechanizm?

bacteria produce pumps in their membrane that remove the antibotic from the interior fo the cell

what is the purpose of peptone in the media

bacteria that can't use the sugars in the media should be able to grow using peptone

Those agents causing temporary inhibition of growth as called what

bacteriostatic agents

what does tetracyclines cause?

causes discoloration of teeth and it's broad spectrum

Antiseptics

chemicals used on living tissue to decrease the number of microbes.

In evaluating the effectiveness of antimicrobial agents, the: .......... are important criteria

concentration, length of contact, and whether it is lethal (-cidal) or inhibiting (-static)

Ionizing radiation

denatures proteins; xrays, gamma rays, Principal effect ionize water into highly reactive free radicals with unpaired electrons that break strands of DNA. Produces peroxides; can be used in meat, kills all growth; pharmacutilcal products; sterilize plastic lab and med equip.

-static means

inhibiting

-cidal means

lethal

what is a disadvantage of the streak plate technique

only organisms in the majority will be isolated

Antimicrobial agents must be matched to specific ......

organisms and environmental condition

Some additional variables to consider in selecting an antimicrobial agent include:

pH, solubility, toxicity, organis material present, and cost

spread plate method

small amount of a previously diluted specimen is spread over the surface of a solid medium using a spreading rod