Biosci 221 Lab Quiz 5

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)

?

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** = 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

(**I**ndole, **M**ethyl Red, **V**oges-Proskauer, **C**itrate)

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

1. Have your complete and detailed stock bacteria notes in front of you
2. Ask your first question --- Keep it broad!
3. Continue to ask simple, but logical, questions.
4. 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