MCB 3020C Microbiology Lab Midterm UCF

framework of microscope

arm and base of microscope

-Always hold the arm and with the other hand support the base

Stage of microscope

Holds the microscope slide in position.

Focusing knob of microscope

-Two focusing knobs located on each side of microscope

-Smaller knob does fine adjustments

-Larger knob does coarse adjustments

Bulb of microscope

Found at the base of microscope

Condensor of microscope

located under the stage

Iris diaphragm of microscope

-located under the stage

-it adjusts the diameter of the cone of the light

rheostat of microscope

used to control the intensity of the light coming from the light source

Microscopy: Illumination

-uses a bright background against which samples will appear dark

Magnification

Total Magnification = Ocular magnification x Objective magnification

Resolution

-measurement of how far apart two objects must be in order for them to be viewed as separate entities

-resolving power of light microscope is 0.2 micrometers

d=(gamma)/(2NA)

Note:

d= smallest distance between two objects which can be seen as separate

gamma= wavelength of light (illumination in nanometers)

NA=numerical aperture (light gathering capacity of lens)

Most dyes have two different components:

Solvent and chromogen

-Chromogen: provides the color and charge to the dyes used for staining

Cationic dyes

BASIC dyes, positively charged chromogen:

.

-methylene blue

-crystal violet

Anionic dyes

ACIDIC dyes, negatively charged chromogin:

.

-acid fuchsin

-Congo red

-Nigrosin

Smearing from broth culture

1. sterilize and cool loop

2. remove the cap of the tube with pinky finger

3. sterilize mouth of tube

4. collect sample

5. sterilize and recap tube

6. make smear, spreading only 1 loopful of culture and spread until maximum thinness

7. sterilize loop

8. Heat fix

Smearing from solid culture (Plate or slant)

1. Add 1 loopful of water

2. remove the cap of the tube with pinky finger

3. sterilize mouth of tube

4. collect sample-just touch the loop to bacteria growth on the plate or slant, mix well in drop of water to suspend

5. sterilize and recap tube

6. make smear

7. sterilize loop

8. Heat fix

Negative stain

is a specialized staining method in which you use an anionic dye to stain the background rather than the actual cells

-negatively charges cell repels the negatively charged chromogen found in the anionic dye

-helpful for identifying morphology and size

-no heat fixing

-used Nigrosin and spreader slide technique

Simple Stain

- is a procedure in which one dye is used to stain all cells

-differential stain

-can be used to identify cell morphology, size, and arrangement

-heat fixed

-uses methylene blue and crystal violet (cationic dyes that carry a positive charge)

-After heat fixing smear, cover with crystal violet for 1min, rinse gently with deionized water, and blot dry with bibulous paper

Stains can be categorized as

Simple, negative, structural, or differential

-coccus(singular) -cocci(plural)

spherical organisms

Bacillus(s)/Bacilli(p)

rod shaped organisms

Spirillium (s)/Spirilla(p)

spiral shape

Pleomorphism

a variety of cell shapes may be seen in a given sample

Diplococci

pairs of cocci

-cells divide in one plane and remain attached after division

Streptococci

chains

-cell division continues to occur in the same plane and cells remain attached to form chains

tetrads (tetracocci)

square of cocci (not cubed)

-cells divide in two planes, the second one perpendicular to the first and remain attached after cell division

Staphylococci

Clusters

-cell division occurs irregularly, cells remain attached after division and a cluster of cells is formed

Sarcinae

cuboidal packets

-cell division occurs in three planes to produce a cube shape

Vibrio

comma shaped

Two of the most common differential stains are

Gram stain and acid-fast stain

What is a gram stain used for?

-differential stain

-developed by Hans Christian Gram who determined what bacteria caused pneumonia

Gram +

thick peptidoglycan layer

-purple

Gram -

Thin peptidoglycan layer

-pink

What is the acid-fast stain used for?

-Acid fast bacteria are Mycobacterium and Norcardia

-Mycobacterium tuberculosis annd Mycobacterium leprae

-differential stain that differentiates bacteria based on the mycolic acid content in their cell walls

-acid fast organisms have higher amounts of mycolic acids in their cell walls than organisms that are not acid-fast.

Acid-Fast Stain: Primary dye, Mordant(if applicable), decolorizer, and counterstain

-Primary Dye: Carbol Fuchsin (Stains both acid fast negative and positive)

-Steaming: makes acid fast bacteria more resistant to decolorization, while acid-fast negative will decolorize

-Decolorizer: Acid Alcohol

-Secondary Dye: Methylene Blue (colors acid-fast negative, blue)

Gram Stain: Primary dye, Mordant(if applicable), decolorizer, and counterstain

-Primary Dye: Crystal violet

-Mordant/binding agent: Gram's Iodine

-Decolorizer: 95% ethanol (makes gram + cells held more strongly together, leaving gram - more porous (gram - become colorless)

-Secondary Dye: Safranin (stains Gram -, pink)Acid

What are/is the endospore forming bacteria?

-genera Bacillus and clostridium

-Bacillus anthracis (cause of anthrax) and Bacillus Cereus (causes food poisoning)

-Clostridium are usually anaerobic. C. Botulinum (causes botulism), C. Tetani (causes gas gangrene), C. Difficile (HAI)

What is the Endospore stain used for?

-is a structural stain that allows you to identify the presence and location of spores in the form of endospores or free spores

-Schaeffer-Fulton Method discovered by Alice B. Schaeffer and Macdonald Fulton

-Spores are stained along with vegetative cells

Endospore Stain: Primary dye, Mordant(if applicable), decolorizer, and counterstain

-Primary Dye: Malachite Green (stains all structure present including free spores, endospores, and vegetative cells

-Steaming: functions to force malachite green into the spores better

-Decolorizer: Water (malachite green is water soluble and does not bind well to bacterial cells); spores will remained stained, while vegetative will release stain and become colorless

-Secondary Dye: Safrinin (stains vegetative cells pink)

A pure culture is one that contains...?

A single bacterial species

Growth Medium

-can be selective or differential and are useful for isolating certain species of bacteria

-in all media there is carbon, nitrogen, inorganic phosphate and sulfur along with trace elements, water, and vitamins

Agar

-a gelatin-like substance derived from the seaweed Gelidium

-first discovered in Japan

-Use of agar in lab is credited to Fanny Hesse

-melts at 85C, remains solid at 32-40C, and most importantly, it can not be degraded by microbes

What are the three different type of isolation methods on a petri dish?

pour plate, Spread plate, and three-zone streak

Pour Plate Method

-not a quantitative technique

-a single loopful of culture is diluted in multiple tubes of molten agar until the sample is diluted enough to allow the growth of individual colonies

-Surface colonies, embedded colonies, and bottom colonies

Spread Plate technique

-useful method when you want to isolate bacteria

-cell suspension sample is serially diluted in some diluent, most often sterile media or sterile saline. Then is added to an agar plate and spread with a spreader.

-only inoculates bacteria on the surface of the agar, and making enumeration of individual of colonies simpler, and more accurate

Three-Zone Streak Plate

-Important for isolating single colonies of bacteria in a mixed or pure sample

-when working with a mixed culture you may isolate using the streak plate method to isolate each bacterial species (Primary streak)

-Following incubation of primary streak, you can select and perform another three-zone streak plate of each species on its own plate to obtain a pure culture of each (Secondary Streak or subculture)

-diluting bacteria by spreading over surface in order to separate cells to encourage growth of individual colonies

Inhibitor

prevent the growth of certain bacteria, which allows you to select for other species that can grow in the presence of these inhibitors

Indicators

detect changes in the media based on a process that has occurred

-either dyes of pH indicators

Complex media

composed of raw materials and are usually undefined, meaning the exact chemical composition is not known

Defined media

Have a known chemical composition and exact amount for each component.

-designed for isolation, selection or differentiation of specific bacteria

Phenylethyl Alcohol Agar (PEA)

-Selective Medium, containing phenylethyl alcohol

-Inhibitor: inhibits growth against Gram(-) bacteria

-Selects for Gram(+) bacteria

-ex. S. aureus

Blood Agar (BA)

-Differential medium composed of a tryptocase soy agar base with 5% sheeps blood

-Differentiates bacteria based on hemolysis patterns

Beta: complete hemolysis of RBC, clearing around bacterial colonies

Alpha: Incomplete (Partial) hemolysis of RBC, discoloration around bacterial colonies as either green or brown

Gamma: No hemolysis of RBC, no change in bacterial colonies

Motility medium

soft-agar medium that is inoculated by stabbing the bacterial sample into the agar deep.

-agar concentration is reduced for better detection of motility

-0.4% agar allows flagellated bacteria to swarm throughout the medium, while non-motile bacteria are confined to stab line.

-Triphenyltetrazolium chloride (TTC) can be added to the medium as an indicator, which produces red color

MacConkey Agar (MAC)

-selective and differential

-used to isolate Gram(-) enteric bacteria and differentiate lactose fermenters from non-lactose fermenters

-Bile salts and crystal violet inhibit the growth of Gram(+) bacteria and some fastidious gram(-)

-Neutral red is the indicator for lactose fermentation

-lactose fermenting bacteria produce acid, which turns the medium reddish-pink. Non-lactose fermenters leave the medium unchanged

Eosin Methylene Blue Agar (EMB)

-Selective & Differential

Selective for: Gram (-) enteric bacteria

-Eosin Y and Mthylene Blue are indicators for lactose fermentation and also acts as inhibitors of Gram(+) bacteria

- Mixed acid fermentation: Strong acid produced by Gram(-) fermenters, makes the medium become a dark purple from strong acid. (E. Coli, leaves a green sheen and also does mixed acid fermentation)

-2,3-butanediol fermenters produce light purple to light pink, which means they produce less acid. (ex. Enterobacter aerogenes exhibits light purple)

-colorless if it does not ferment lactose, and no growth for Gram(+)

Viable cells

those which are able to reproduce when cultured

-bacterial suspension is serially diluted and then a small sample from each dilution is spread on the surface of an agar plate

Dilution Factor equation

Dilution = A/(A+B)

A=volume of sample added to a blank (mL)

B=Volume of blank

ex. Dilution = (0.1mL/(0.1mL+9.9mL) =

0.1mL/10mL=1:100 or 10^-2

Plate Dilution equation

Plate dilution = (mL plated)/(1mL)

Final dilution = Tube dilution x plate dilution

The original cell density of the sample is calculated in...?

Colony forming units

-multiplying the # of colonies growing on the plate by the inverse of the final dilution factor

Plate counts below 30 colonies are considered...?

too few to count (TFTC)

Plate counts above 300 colonies are considered...?

Too numerous to count (TNTC)

Exoenzymes

Extracellular enzymes produced by bacterial species

-can be hydrolytic enzymes

Hydrolytic enzymes

break down large substrates into smaller components by the addition of water so they can be transported into the cell for use in cellular metabolism

Lipase Agar

-Tests for Lipase: is an exoenzyme used by bacteria to hydrolyze fats into glycerol and fatty acids

production of lipase

-Indicator: Spirit Blue is an indicator for hydrolysis

-Lipase reagent: mixture of tributyrin(a fat) and polysorbate 90 that emulsifies fats to make them more soluble in water

-Positive Result: A dark blue halo producing an intensification of the blue color around bacterial growth indicates the production of lipase

-Negative result: clearing around bacterial growth and lack of blue.

Milk Agar

-Test for Caseinase: is an exoenzyme secreted by bacteria that hydrolyzes casein into amino acids

Production of caseinase

Indicator: A clear zone around bacterial growth

Reagent: Milk Agar contains skim milk, which contains lactose, casein, peptone and agar.

Positive Result: Clearing around bacterial growth which is caused by caseinase

Negative Result: No clearing around bacterial growth

Starch Agar

-Tests for Amylase: is an exoenzyme which breaks down starch into glucose, a simple sugar, that can be transported through the cell's membrane

-Indicator:Iodine will complex with any starch remaining on the plate

-Reagent: Starch agar contains beef extract, soluble starch, and agar.

Positive Result: halo (clearing) around the bacterial growth after the addition of iodine is a positive result for the presence of amylase

Negative result: Dark or purple color around colony is negative

Litmus Milk

-Tests for Fermentation of lactose and production of caseinase

-Indicator: Litmus, pH indicator which is blue under alkaline conditions and pink under acidic conditions

-Reagent: Litmus milk contains skim milk and litmus

Positive Results:

-Acid Production

-Alkaline production

-Curd

-reduction(redox)

-proteolysis

Note: An uninoculated tube is purple (control)

Litmus Milk: Acid production

Microbes ferment lactose, acids are produced that reduce the pH of the litmus milk solution, turning the litmus pink

-Varying by degrees of acid production, from weak to strong, which can be seen by varying shades of pink.

-Compare with uninoculated control tibe

Litmus Milk: Alkaline production

In the absence of lactose fermentation the bacteria may instead deaminate peptones which results in the release of ammonia (NH3) into the solution

-An increase in NH3 raises the pH of the litmus milk, turning solution blue

-Peptone deaminate is an oxygen-dependent process, so the color change may be limited to the top of the tube.

Litmus Milk: Curd

If enough acids are produced as a byproduct of lactose fermentation, the milk proteins may become denatured, which leads to the formation of a hard curd

- some curds completely solidify the milk or may incompletely curdle, leading to a lumpy consistency

Litmus Milk: Reduction (redox)

-Litmus milk only functions as a pH indicator if it is in an oxidized redox state; under reducing conditions, litmus loses its color

-A band of color will persist at the top of the media, where oxygen can penetrate and reoxidize the litmus indicator

Litmus Milk: Proteolysis

-some bacteria can produce the exoenzyme caseinase that can break down the protein casein, which gives milk its white color.

-If casein is broken down (proteolyzed), the milk will turn clear

Fermentation

-an energy yielding process where a nutrient molecule is broken down without net oxidation

-differential media can be used to help identify fermentation

Phenol Red Broth (Sugar fermentation broth)

-Fermentation of a given sugar (mannitol, lactose, or glucose)

-It will turn cerise(hot pink) if the bacteria does not use sugar and utilizes peptone instead, which releases NH3 into medium

-Indicator: Phenol Red: pH indicator &Durham tube-detects gas production.

Reagent: Phenol red broth, with the pH indicator, phenol red, a single sugar (mannitol, lactose, or glucose), and a durham tube

-Positive result: Medium turns yellow if the sugar is fermented, the phenol red will turn yellow in the presence of acidic byproducts. If gas is present, bubble will be in durham tube. Medium turns pink(cerise): If the bacteria present doesn't use the sugar provided, they may instead utilize peptones, which leads to the release of NH3, and therefore increases the pH

-Negative result: No change in phenol red broth, red remains the same and no gas in durham tube

Kilger's Iron Agar (KIA)

Tests for: Lactose fermentation, glucose fermentation, and sulfide production by the bacterial enzyme, Cysteine desulfhydrase

Products: Fermentation of glucose only, both lactose and glucose, or no fermentation

Indicator: Phenol Red is the pH indicator, while ferric ammonium citrate is the indicator of sulfide production

Reagent: Medium contains two sugars: 0.1% glucose, 1% lactose, along with 1% peptones. Also contains beef and yeast extract

Positive Results:

Lac-,Glu-,H2S-=control

Lac+,Glu+,H2S-=complete yellow

Lac+,Glu+,H2S-=small pink at tip of slant and yellow beneath

Lac-,Glu+,H2S-=Pink all the way down slant top and yellow beneath

Lac-,Glu+,H2S+=Darkp purple/black completely

Gas production= cracking or lifting

Negative result:No fermentation, organism is respiratory (pink)

KIA: Lactose fermentation positive

acid can be formed from both the fermentation of glucose (0.1%) and lactose(1%).

-lactose is a disaccharide that can be hydrolyzed to glucose from fructose by the enzymes Beta-galactosidase, if an organism can ferment lactose, by definition it can also ferment glucose.

-large amounts of acide produced by lactose and glucose fermentation overwhelm any alkaline products of peptone degradation, leaving entire tube yellow.

KIA:Lactose fermentation negative

-If only glucose is fermented, then the amount of NH3 produced by breakdown of peptone will exceed the amount of acid produced by glucose fermentation (1.0%>0.1%)

-The breakdown of peptone will only take place in the presence of oxygen, therefore deamination of peptones will keep only the upper slant at a neutral/alkaline pH (pink). The butt of the tube will remain yellow due to glucose fermentation.(O2 can not penetrate far enough into the butt of the tube for peptone deamination, to reverse acidification.

KIA: Reversion

-Some lactose fermenting bacteria produce weaker, less stable acids that can be oxisizes into neutral end products. This can lead to reversion of part of the slant back to neutral/alkaline pH.

-observed with enterobacter aerogenes, which uses 2,3-butanediol pathway for lactose fermentation

-KIA is ideally read at about 18hrs after inoculation. Inoculations older than 18hrs may give incorrect readouts

-Small pink on tip of slant, butt is yellow

KIA:Sulfur Reaction

-Reduction of sulfur produces H2S, which combines with iron in the media to form black precipitate (ferrous sulfide).

-Ferrous sulfide precipitate will obscure color in the bottom of the tube.

-H2S will only form as a byproduct of sulfur reduction in an acidic environment

Peptides for energy

bacteria can catabolize polypeptides and perform respiration

deamination

removes amino group

-in phenylalanine slant, it tests for an enzyme that removes the amino group from phenylalanine, through deamination

Decarboxylation

-an enzyme removes the carboxy group from the amino acid, releasing CO2 , and the corresponding amine, which increase the pH of the media

Desulfhydrase

removes hydrogen sulfide H2S from the amino acids that contain sulfur, such as the amino acid cysteine and methionine

Methyl Red Test

Tests for: mixed acid fermentation pathway

Products:stable acids that came from the breaking down of glycolysis to pyruvate.

Indicator: Methyl red

Positive test: pH<4.4 = remain vibrate red color

Negative tests: pH 4.4-6.2=orange color& pH>6.2 = yellow

Voges-Proskauer Test

Tests for: organisms that produce 2,3-butanediol as a final product through the fermentation of glucose

Produces: acid products produced by glucose fermentation which are quickly converted to acetoin and then 2,3-butanediol

Indicator: acetoin, NO pH indicator

REAGENTS: AS FOLLOWS

Barritt's Reagents or VP reagent A/B

-VP reagent A (alpha-naphthol):works as a catalyst that will intensify the color and increase sensitivity of the rxn.

-VP reagent B (KOH): used as an oxidizing reage to change acetoin to diacetyl, which then reacts with guanidine nuclei from the peptones in order to produce red color

Positive test: Red color is positive for VP test

Negative test: Negative if remains yellow

Urease

-Tests for Urease,distinguishes members of genus Proteus from nonlactose fermenting members of the family Enterobacteriaceae

*degrades Urea->NH3 +CO2

*increases pH more alkaline

-urease removes NH3 from urea

Indicator: Phenol red

Positive Result: pH>8.4= cerise pink due to the production of ammonia

Negative Result: pH=6.8, yellow for acidic, red for neutral

Gelatin Hydrolysis test

-Test for: exoenzyme gelatinase, used to determine bacteria that produce the exoenzyme gelatinase, which liquefies gelatin media

-Positive results: Positive tubes will liquefy

-Negative Results: Negative tubes will solidify as gelatin cools

bacillus species, staph a., and some enterobacter.

Simmons Citrate Test

-Tests for: utilization of citrate as sole carbon source for growth

[Note: to distinguish enterobacter aerogenes, which can utilize citrate as a carbon source and E. Coli can not.]

-Produces: Citrate permease, which brings the citrate inside the cell, and a citrate lyase, which breaks down citrate into pyruvate which then can be reduced during fermentation

-Indicators: Bromothymol Blue

-Positive Results: pH>7.5 bromothymol blue will turn media into royal blue

-Negative Results: Green tubes with no sign of growth indicates that the inoculated organism cannot utilize citrate as its sole source of carbon

Phenylalanine Test

-Tests for: production of phenylpyruvic acid, deaminase enzyme.

Note: used to distinguish between bacteria in the Enterobacteriaceae

Products: Phenylpyruvic acid -->Certain organisms are able to convert phenylalanine into phenylpyruvic acid & ammonium using the enzyme phenylalanine deaminase.

-Indicator: 10% Ferric chloride (FeCl3) is added to the slant to indicate the production of phenylpyruvic acid (PPA)

-Reagent: 10% FeCl3

-Positive Result: In the presence of PPA, ferric chloride reacts with PPA and creates a deep green color

-Negative Result: No green

Tryptone Broth Test/Indole test

-Tests for: Indicates production of tryptophanase, which converts amino acid tryptophan into indole, pyruvate, and ammonium

-Produces: Indole production through DMABA

-Indicators: Kovac's reagent (p-dimethyl-aminobenzaldehyde, DMABA)

-Reagents:Kovac's reagent (p-dimethyl-aminobenzaldehyde, DMABA)--> nonpolar and extracts indole at top of tube.

*Indole +kovac = cerise

-Positive Results: Cerise at top

-Negative Results: No cerise at top, instead yellow

SIM

-Test for: Sulfide, Indole, and motility

-Products: As follows

sulfide-detects for production of cysteine desulfhydrase, produces black precipitate

Indole-detects production of enzyme tryptophanase, produces cerise color, which indicates production of indole

motility-identify if motile spread throughout tube

-Reagent: Indole-kovac's reagent

-Positive results:

black precipitate-sulfide reduction.

Cerise color- indole production

Cloudiness-indicates motility

Oxygenic respiration or aerobic

If oxygen is used

Anaerobic respiration

if something other than O2 is used for respiration, such as nitrate or CO2.

Catalase Test

Tests for: production of catalase -> converts H2O2 into H2O and O2

-Products: water and oxygen

Indicator: Bubbles

Reagent: 3% Hydrogen peroxide(H2O2)

Positive Results: If bubbles are present, organism can produce catalase

Negative Result: no bubbles, no production of catalase

Stapha. &L. Lactis

Oxidase Test

-Tests for: the presence of the enzyme Cytochrome C oxidase

-Indicator: dimethyl-p-phenylenediamine, colorless indicator

-Positive Results: Culture turns blue if cytochrome C oxidase is present

-Negative Results: colorless

Nitrate Reduction Test

-Test for: Nitrate Reductase, which reduces nitrate to nitrite

[Note:distinguishing members of the family Enterobacteriaceae from other gram(-) bacteria]

-Indicators: production of nitrate reductase is tested by two reagents, Nitrate 1 (sulfanilic acid) and Nitrate 2(dimethyl-alpha-naphthylamine).produces a brick red color to detect the presence of NO2, after 2min

-Reagents: two reagents, Nitrate 1 (sulfanilic acid) and Nitrate 2(dimethyl-alpha-naphthylamine)

-Positive Results: -After nitrate 1&2 are added to NO3, if it turns brick red, then it is positive and was reduced to NO2. If it was colorless, then zinc is added and should remain colorless, which means it was reduced all the way to N2.

-Negative Results: If after the addition of zinc, it turns red, then it is negative, because NO3 is still present in tube

P.aeruginosa, L. Lactis, B. cereus

What plating methods can be used to obtain isolated colonies?

Pour plate method can be used to isolate colonies through the process of dilution

What does a viable plate count do?

-a bacterial suspension is serially diluted and then a small sample from each dilution is spread on the surface of an agar plate.

-measures the number of pathogens per milliliter of blood,urine or other fluids

Regarding the viable plate count, what is the countable range?

30-300

How can media be classified (what they do or how nutrients are provided)?

Both, they are classified by what they do and how nutrients are provided.

-there are for types of media based on purpose: general purpose,selective,differential, and combination(selective and differential)

-2 types depending on composition: complex or defined

Selective media vs differential media

Selective Media:

-contain an inhibitor, which will prevent the growth of certain bacteria

Differential Media:

-contains indicators that detect change in the media

some media have both such as EMB and MacConkey agar