Control of Microbial Growth (Antimicrobial Sensitivity, Use Dilution, Antiseptic Testing), Physiological Testing, and Identifying Unknowns
Sterilization
Destruction of ALL living cells, spores, and/or viruses
Disinfection
The disinfection process inactivates most microbes on the surface of a fomite (inanimate items which may harbor microbes and aid in disease transmission) by using antimicrobial chemicals or heat
Meaning… the removal of disease-causing microorganisms from inanimate surfaces (does not kill all organisms)
Disinfectants
Disinfectants should be fast acting, stable, easy to prepare, inexpensive, and easy to use
Ex.:
Natural disinfectant = vinegar
Chemical disinfectant = chlorine bleach or products containing chlorine
Antiseptics
Antiseptics are antimicrobial chemicals safe for use on living skin or tissues
Ex. hydrogen peroxide and isopropyl alcohol
Antisepsis
The process of applying an antiseptic is called antisepsis
The removal of disease-causing microorganisms from living tissues
Use Dilution Method - what are the purposes of the steps and the various tubes for incubation
The use-dilution test is commonly used to determine a chemical’s disinfection effectiveness on an inanimate surface in clinical settings
For this test, a cylinder of stainless steel is dipped in a culture of the targeted microorganism and then dried. The cylinder is then dipped in solutions of disinfectant at various concentrations for a specified amount of time. Finally, the cylinder is transferred to a new test tube containing a fresh sterile medium that does not contain disinfectant, and this test tube is incubated. Bacterial survival is demonstrated by the presence of turbidity in the medium, whereas killing the target organism on the cylinder with the disinfectant will produce no turbidity
Understand the concept of "control"
To prevent the spread of human disease, it is necessary to control the growth and abundance of microbes in or on various items frequently used by humans
Any significant results?
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Antibiotic
chemicals derived from microorganisms that inhibit or kill bacteria
Chemotherapeutic agent
synthetic drugs that serve the same purpose as an antibiotic
In vitro
(of a process) performed or taking place in a test tube, culture dish, or elsewhere outside a living organism
In vivo
(of a process) performed or taking place in a living organism
McFarland Standards
The MacFarland standard method employs a prepared test tube of specific turbidity with which you can prepare a broth of the bacterium to be tested to the same turbidity.
This helps ensure that a consistent amount of bacteria is plated.
Mueller Hinton agar (and its importance)
a non-selective and non-differential growth medium that is commonly used for microbial sensitivity testing
used for clinical testing
helps identify microbes
loose agar meaning when you place antibiotics on the media it's allowed to diffuse or seep in, allowing for antibiotic susceptibility tests to be conducted accurately
History of important scientist Dr. Jane Hinton - what were her important contributions?
helped develop Mueller-Hinton Agar
still used in laboratories today to determine antibiotic resistance among bacteria
Determination of:
Susceptibility/Sensitivity
Intermediate
Resistance
The antibiotics are prepared specifically for the antimicrobial sensitivity test by infusing known amounts into standard paper disks. As bacteria begin to grow on a plate, the antibiotics are diffusing out into the agar. If the bacteria are sensitive to the antibiotic, at some point their growth will be inhibited and a zone of no growth (zone of inhibition) will be apparent around the disk. The diameter of the zone is measured and the size of the zone determines whether a bacterium is determined Sensitive (S), Resistant (R), or Intermediate (I) to the antibiotic.
The size and interpretation of the zone is specific to each bacteria-antibiotic pair and is referenced in a chart.
Novobiocin results of Staphylococcus saprophyticus and Staphylococcus epidermidis
S. saprophyticus is resistant
S. epidermis is susceptible
Mode of Action of the antibiotics listed in our handout (looked up in the chart in our text - page number given in the handout)
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MacConkey agar (MAC)
Purpose: to differentiate between lactose fermenting G- bacteria and lactose non-fermenting G- bacteria
Medium: crystal violet and bile salts
Type of Medium: selective and differential
+ results = pink colonies
- results = no color change
significant specific ingredients:
reagents/indicators:
crystal violet and bile salts inhibit Gram (+) bacteria
and neutral red dye stains microbes fermenting lactose (and thereby decreasing the pH) a pink color.
specific directions (if any): streak agar in a straight line and incubate
significant results:
Columbia Naladixic Acid agar (CNA)
Purpose: for the isolation of G+ cocci
Medium: contains the antibiotics colistin and nalidixic acid
Type of Medium: selective and differential
Growth (G) = clearly visible, good growth
Weak growth (WG) = very little growth
No growth (NG) = no visible growth
significant specific ingredients:
reagents/indicators: colistin and nalidixic acid inhibit G- bacteria
specific directions (if any): streak agar in a straight line and incubate
significant results:
Catalase
Purpose: to detect the production of the enzyme, catalase
Medium: TSA
Type of Medium: differential
+ results = bubbling due to the release of oxygen via catalase
- results = no bubbling
significant specific ingredients:
reagents/indicators: 3% Hydrogen Peroxide (H2O2)
specific directions (if any): apply 3% hydrogen peroxide to growth from a TSA plate
significant results:
Oxidase
Purpose: to detect the production of the enzyme cytochrome c oxidase
Medium: use growth from a TSA plate or slant
Type of Medium: differential
+ results = color change to purple within about 30 sec
- results = no color change or a change after more than 30 sec
significant specific ingredients:
reagents/indicators: oxidase dry slides; used after growth on TSA.
specific directions (if any): using a sterile wooden stick, pick a colony of bacteria from a TSA plate or slant and touch an area on one section of the dry slide
significant results:
Methyl Red test (MR)
Purpose: to determine mixed acid fermentation (lactic, acetic, formic, etc)
Part of the IMViC tests.
Medium: MRVP broth--buffered peptone glucose broth
Type of Medium: differential
+ results = red
- results = yellow
weak + = orange
significant specific ingredients:
reagents/indicators: methyl red (added AFTER incubation)
specific directions (if any): broth is inoculated and incubated. After incubation, add 5 drops of Methyl Red indicator, do not shake the tube, and read the results immediately
significant results:
Voges Proskauer test (VP)
Purpose: to detect the production of acetoin or butanediol from the fermentation of glucose in the broth
Part of the IMViC tests.
Medium: MRVP broth-- buffered glucose peptone broth
Type of Medium: differential
+ results = red layer at the top in 10 minutes (earliest detection), progressing downward
- results = no red color, disregard any copper or brownish-purple color
significant specific ingredients:
reagents/indicators: Barritt’s reagents A and B added AFTER incubation
specific directions (if any): inoculate broth and incubate. After incubation add 20 drops of Barritt’s Reagent A and 20 drops of Barritt’s Reagent B. Vortex at frequent intervals and allow the reaction to develop for up to 1 – 2 hours.
significant results:
Phenol Red Broths - Lactose, Dextrose (Glucose), Sucrose
Purpose: to distinguish carbohydrate fermenters from non-fermenters & to detect and distinguish the different, specific carbohydrates by the products formed
Medium: 0.5% to 1% carbohydrate (dextrose/lactose/sucrose) broth, peptone, with phenol red and an inverted Durham tube for detection of gas.
Type of Medium: general purpose differential test media
+ results = turns from red to yellow
- results = no color change
Gas production (+) = bubble trapped in an inverted Durham tube
No gas production (-) = no bubble trapped in an inverted Durham tube
significant specific ingredients:
reagents/indicators: phenol red as a pH indicator to indicate acid production
specific directions (if any): inoculate tubes and incubate
significant results:
AG = acid with gas production
A = acid, no gas
(-) = negative for acid and gas
Simmon's Citrate test
Purpose: to determine an organism’s ability to use citrate as the sole source of carbon
Part of the IMViC tests.
Medium: Simmons Citrate Agar- contains sodium citrate as the sole carbon source, mineral salts, and pH indicator Bromothymol blue
Type of Medium: selective and differential
+ results = color change from green to blue
- results = no color change
significant specific ingredients:
reagents/indicators: Bromothymol blue is a pH indicator
specific directions (if any): streak slant, cap loosely, and incubate.
significant results:
Starch hydrolysis
Purpose: to detect the production of the enzyme amylase
Medium: Starch Agar plates (1% starch)
Type of Medium: differential
+ results =Â clear zone around growth
- results = no zone
significant specific ingredients:
reagents/indicators: Gram’s Iodine
specific directions (if any): streak agar in a straight line and incubate. After incubation, add Gram’s iodine, dropwise, sparingly, just to cover growth and surrounding area on the medium.  Let the plate sit for a few minutes for the reaction to develop
significant results:
Casein hydrolysis (Skim Milk)
Purpose: to detect the production of the enzyme casease
Medium: Skim Milk Agar
Type of Medium: differential
+ results = clear zone around growth
- results = no clear zone
significant specific ingredients:
reagents/indicators:
specific directions (if any): streak agar in a straight line and incubate
significant results:
Urease production
Purpose: to detect the production of the enzyme urease
Medium: urea broth
Type of Medium: differential
+ results = red or bright pink color
- results = yellow color
significant specific ingredients:
reagents/indicators: Phenol Red
specific directions (if any): inoculate urea broth and incubate
significant results:
Gelatin Liquefaction
Purpose: to determine the production of gelatinase
Medium: Nutrient Gelatin Deep (12- 15% gelatin)
Type of Medium: differential
+ results = Liquefaction (after refrigeration)
- results = Gels when refrigerated, no liquefaction
significant specific ingredients:
reagents/indicators:
specific directions (if any): deep stab inoculation and incubate. After incubation, refrigerate for 1 hour before reading.
significant results:
Sulfide, Indole, Motility (SIM) Medium
Purpose: to differentiate G- rods
Medium: contains casein peptone, ferrous ammonium sulfate, sodium thiosulfate, and agar
Type of Medium: selective and differential
+ results =
Sulfide (+) = blackening of the media
Indole (+) = pink to red color observed on the surface after the addition of Kovac’s reagent
Motility (+) = growth observed beyond the stab line (cloudy, turbid)
- results =
Sulfide (-) = no blackening of the media observed
Indole (-) = yellow color observed on the surface after addition of Kovac’s reagent
Motility (-) = no growth observed beyond stab line (crisp solid inoculation line visible)
significant specific ingredients:
reagents/indicators:
ferrous ammonium sulfate and sodium thiosulfate are present to detect hydrogen sulfide (H2S) production
The agar in the medium creates a semi-solid environment for motility to be visualized
Kovac’s reagent (added AFTER incubation) will detect indole production
specific directions (if any): inoculate the media using your needle, stabbing down the center of the media in the tube
significant results:
IMViC tests
(Indole, Methyl Red, Voges-Proskauer, Citrate)
a battery of tests to help identify enterics and other GNRs
First steps in identifying Unknowns
inoculating? determine gram stain and morphology?
How to set up a dichotomous key
Have your complete and detailed stock bacteria notes in front of you
Ask your first question --- Keep it broad!
Continue to ask simple, but logical, questions.
Now continue asking questions, getting more and more specific until you narrow it down to one organism
Understanding how to use the information you have, what the limitations are, how not to make assumptions
Culture characteristics can look different due to age, dryness, competition, etc.
Compare your results with your group members and other groups to check for discrepancies.
Be 100% certain before excluding anything, even if only slightly unsure, do not exclude!
Always ask: “Could it be something else?”
What are all 18 stock bacteria and their Gram reaction and morphology?
(photo)
Are there tests useful in identifying each one of the bacteria?
Citrate test → E.coli