Broken glassware, wooden cotton swabs, disposable inoculation loops, etc. MUST be placed in the correct ______ container. Sterile in the _______ container, and used in the ________ container.

- "sharps"

- standard sharps

- biohazard sharps

Define the following: magnification, resolution, contrast

- degree of enlargement of a specimen

- measure of the clarity of an object

- difference in brightness between light and dark areas of a specimen

Calculation of LM. Name the 4 lenses and the magnifying power associated with each. Name the total magnification of each lens (including ocular).

- Scanning Objective, 4X = 40X

- Low Power Objective, 10X = 100X

- High Power Objective, 40X = 400x

- Oil Immersion Objective, 100X = 1000X

Describing common colonies.

Elevation, form, size, surface, texture, color, opacity, margin

- elevation: side view of a colony

- form: basic shape of a colony

- size: diameter of the colony

- surface: smooth, rough, shiny, wrinkled

- texture: dry, moist, mucoid, brittle

- color: white, buff, red, purple, orange, etc.

- opacity: transparent, opaque, translucent, cloudy, etc.

- margin: the edge of the colony, undulated, lobate, curled

Describe the difference between the 4 quadrants of a streak plate.

- 1st: heavy confluent (merged) growth

- 2nd: heavy growth

- 3rd: light growth

- 4th: discrete (individual) colonies

What is the Smear technique? What are the steps you follow?

- spread a thin film made from a liquid suspension of cells on a slide

- allow the slide to air dry completely

- heat fixing: heat the slide gently (not in simple negative staining)

What are the important functions of heat-fixing?

- kills the cells (will not contaminate handler's fingers)

- secures the specimen to the slider (caramelization of PGs & LPS is sticky)

- preserves cellular components in a natural state with minimal distortion

- coagulates their proteins for better staining

Explain Basic Dyes [Ch+]OH-

- have positively charged (cationic) chromogen

- are attracted to the negative charges on the bacterial surface

- color the cells

- are applied to bacterial smears that have been heat-fixed

- Ex: methylene blue, crystal violet, and safranin

Explain Acidic Dyes [Ch-]H+

- have negatively charged (anionic) chromogen

- are repelled by negative charges on the bacterial surface

- color the background, not the cells

- are applied to bacterial smears that have NOT been heat-fixed

- Ex: nigrosin, eosin, and acid fuchin

Name the categories for simple staining, differential staining, and special staining

- Simple: positive; negative

- Differential: Gram; acid-fast; spore

- Special: capsule; flagella

Simple Stain

- used to highlight and stain the entire organism

- uses one color dye

- you can see the shape, some basic structures, and relative size

Differential Stain

- uses 2 colored dyes

- second dye is called a counterstain (usually it's a contrasting color to the principal stain)

- used to contrast two cell types and indicate cell parts (ex: Gram stain, endospore stain)

Simple Staining (II)

- has limited use in microbiology

- allows researchers to identify only the size, shape, and arrangement of the bacterial cells

- CAN'T provide information about their nature

Differential Staining (II)

- provides the same information that a simple stain provides (size, shape, arrangement)

- also provides information about the nature (structural composition and, to some extent, physiology) of the bacterial cells

----differences in the composition of the cell wall between different species of bacteria: G+, G-, mycobacteria (this is crucial to the clinician as it plays a role in how we are able to treat infections caused by these bacteria)

----ability of bacteria to produce endospores

What is the purpose of the primary dye in differential staining?

- provides contrast between your cells and the slide

- helps you quickly identify cells/structures that are "positive" for the staining technique you are performing

What is a mordant, and why do we use it in differential staining?

- it is a fixative agent

- makes sure the primary dye adheres well to the cells/structures that are meant to appear "positive" for the staining technique you are performing

- can be a chemical compound or something as simple as heat

What is the purpose of a decolorizing agent?

- selectively removes the primary dye from cells/structures whose physical structure is different from those cells/structures previously stained with the primary dye and, therefore, should be "negative" for the particular staining technique. Unfortunately, this also causes those cells to now appear more or less colorless

- most crucial step; failure to do this will result in cells that all appear the same color, eliminating the advantage of performing a differential stain

What is the counterstain?

- provides a different color to cells that should be "negative"

Describe a Gram-positive bacteria.

- produce a thick, rigid cell wall composed entirely of peptidoglycan (PG), combined with other compounds such as teichoic acids to give them additional strength and stability

Describe Gram-negative bacteria.

- produce much thinner cell wall composed of PG

- they have an additional layer of lipopolysaccharide (LPS) that is located beyond the peptidoglycan layer and is responsible for the virulence of many Gram-negative bacteria

What is the Gram Stain procedure?

1. Crystal violet - added to the cells in a smear; stains them all the same purple color

2. Gram's iodine - the mordant is added; this is a stabilizer that causes the dye to form large complexes in the PG meshwork of the cell wall. the thicker G+ cell walls are able to more firmly trap the large complexes than those of G- cells

3. Alcohol - application of alcohol dissolves lipids in the outer membrane and removes the dye from the PG layer (only in the G- cells)

4. Safranin - because G- bacteria are colorless after decolorization, their presence is demonstrated by applying the counterstain safranin in the final step

General media

- permit the growth of all organisms regardless of type or nutritional peculiarities

Selective media

- contain one or more inhibitory agents designed to prevent the growth of some microbes while favoring the growth of others

Differential media

- contain chemical indicators along with additional nutrients that may or may not be utilized by some species of microbes

Phenylethyl Alcohol Agar (PEA)

- Selective Agent: phenylethyl alcohol

- favors growth of G+

Columbia CNA w/ 5% Sheep Blood Agar

- Selective Agent: Colistin and Nalidixic Acid (CNA)

- favors growth of G+

- Differential Agent: 5% blood

- Indicator: hemolytic activity 

---- beta hemolysis: complete degradation

---- alpha hemolysis: incomplete degradation

---- gamma hemolysis: no degradation

-Useful in identifying the pathogenic Staphylococcus, Streptococcus, and Enterococcus

Mannitol Salt Agar (MSA)

- Selective Agent: 7.5% NaCl

- favors the growth of halo-tolerant Staphylococcus

- Differential Agent: Mannitol

- Indicator: Phenol Red

---- Mannitol fermentation will produce a yellow color change as the pH of the media drops below 6.8

---- Indicative of Staphylococcus aureus

MacConkey Agar (Mac)

- Selective Agents: Bile Salts and Crystal Violet

- favors growth of G-

- Differential Agent: Lactose

- Indicator: Neutral Red

---- Lactose fermentation causes a shift from colorless to hot pink as the pH drops below 6.8

---- indicates a possible coliform (universally present in large numbers in the feces of warm-blooded animals; indicator of sanitary quality of foods)

Eosin Methylene Blue Agar (EMB)

- Selective Agents: Eosin and Methylene Blue

- favor growth of G-

- Differential Agent: Lactose and/or Sucrose

- Indicator: Eosin / Methylene Blue

---- Lactose and/or sucrose fermentation will produce a color change caused by interactions with the dyes (Heavy acid = dark colonies; Light acid = pink colonies)

---- Fermentation = possible coliforms

Hektoen Enteric Agar (HE)

- Selective Agent: Bile Salts

- favors growth of G-

- Differential Agents: Lactose, Sucrose, and Salicin

- Indicators: Bromthymol Blue and Acid Fuchin

---- sugar fermentation causes a pink to orange color change and is a negative result for Salmonella or Shigella

- Differential Agent: Sodium Thiosulfate

- Indicator: Ferric Ammonium Citrate

----H2S production causes a black precipitate

---- Possible Salmonella or Shigella

Aerotolerance

ability or inability of microbes to survive in the presence of O2

Fluid Thioglycollate Medium (FTM)

- used to determine the oxygen requirements of microorganisms

- consumes O2 and permits the growth of even obligate anaerobes

- this, combined with the diffusion of oxygen from the top broth, produces a range of oxygen concentrations in the medium along its depth

Psychrophile

- grow only below 15°C

Psychrotroph

- adapted to grow between 0°C and 30°C

Mesophile

- adapted to grow between 15°C and 45°C

Thermophile

- adapted to grow above 45°C

Extreme Thermophile

- grow best above 80°C

Resazurin

- dye that turns pink when it is oxidized by oxygen; shows how far O2 penetrates into medium

Obligate

- strict

- existing in a very narrow niche

Facultative

- the ability to exist in a wide range of conditions

Obligate Aerobes

- grow at the top of the medium, where O2 is present in high concentration

Obligate Anaerobes

- grow below resazurin dye, at the bottom part of the test tube, where there is little to no O2

Facultative Anaerobes

- grow throughout the test tube since they can:

---- respire aerobically (by reducing O2)

---- respire anaerobically (by reducing sulfur or nitrate instead of oxygen)

---- ferment available substrate

Microaerophiles

- grow in the middle or upper middle part of the medium since they only survive in environment containing lower than atmospheric levels of O2

Starch Hydrolysis Test

- Differential Agent: Starch (amylose + amylopectin are α-amylose polymers)

-- Used to identify the presence of α-amylase and oglio-1, 6-glucosidase

-- enzymes hydrolyze starch releasing individual glucose

- Indicator: Iodine that is added after incubation has occurred

-- clearing around a colony indicates absence of starch (it was digested w/ amylase)

-- presence of residual starch is indicated by brown color (or blue-black)

Casein Hydrolysis Test

- Differential Agent: Milk

-- used to identify the presence of casease which hydrolyzes casein (the primary protein in milk)

- Indicator: clearing of the "white" color produced by the milk protein, casein

Lipid Hydrolysis Test

- Differential Agent: Tributyrin Oil (triglyceride)

-- used to identify the presence of lipases that hydrolyze lipids or triglycerides for transport into cells

-- Converts the fatty acids into acetyl-CoA through beta-oxidation

- Indicator: clearing of the "cloudiness" originally produced by the presence of oil in the agar

DNA Hydrolysis Test

- Differential Agent: DNA

-- identifies organisms that can produce deoxyribonuclease (DNase)

- Indicator: Methyl Green Dye

-- dye forms a complex with DNA to give a blue-green appearance

-- Hydrolysis (elimination) of the DNA removes the dye causing the media to turn clear since nucleotides don't bind to dye

Bile Esculin (BESC) Test

- Selective Agent: Bile

-- Inhibits all Gram (+) bacteria EXCEPT enterococci and streptococci group D

- Differential Agent: Esculin 

-- identifies streptococci and enterococci that hydrolyze esculin to esculetin

- Indicator: Ferric Ammonim Citrate

-- esculetin reacts with ferric citrate to form a dark color

-- esculin → esculetin → glu + FAC (Fe) → black precipitate