-Growth occurs throughout the container and has a dispersed cloudy or flaky appearance.

Semi-Solid -At room temperature they exhibit a Clot-like consistency because they contain an amount of solidifying agent (agar or gelatin) that thickens them but does not produce a firm substrate. This media is used to determine the motility of bacteria and to localize a reaction at a specific site. semisolid media can be used to test for physiological characteristics used in identification ( hydrogen sulfide production and indolereaction).



Solid
-Firm surface, due to addition of agar of gelatin, on which cells can form discrete colonies and are advantageous for isolating and culturing bacteria and fungi
Two forms:
-liquefiable solid media, reversible solid media, contain a solidifying agent that changes its physical properties in response to temperature. The most widely used media because it is solid at room temperature and melts.
-Agar-complex polysaccharides isolated from the red agla Gelidium.
-Role of Fannie Hess.
-non liquefiable solid media have less versatile applications than agar media because they do not melt.
-Reversible (liquid-solid, solid-liquid)
-Not readily digestible to microbes
-If the medium contains 1-5% agar, it uses the word agar in its name
-Other solid mediums
-Rice grains
-Cooked meat
-Potato slices
-Egg

Chemical Content of Media



Synthetic:

-The medium is chemically defined. This means that it only contains pure organic and pure inorganic compounds that are specified by an exact formula.The content varies little between sources.
-Minimal media, for fungi, contain only a few essential compounds like salt and amino acids, other types of synthetic media are useful in research and cell culture when the exact nutritional needs of the test organisms are known.
Non-synthetic:

-The media contains one or more non-chemically definable component. Examples of non-synthetic medium include extracts from animals, plants, meat extracts, milk, serums, blood, Peptone or yeast.
Examples:
1.Blood
2. serum
3. meat extracts
4. milk
5. serums-high in vitamins, minerals, proteins, and other organic nutrients
6. yeast extracts
7. soybean digests
8. peptone-partially digested protein, rich in amino acids, a source of carbon or nitrogen

Functional Categorization of Media


General Purpose:
-Depending upon what is added, a microbiologist can fine tune a medium for nearly any purpose, only a few species of bacteria or fungi cannot yet be cultivated artificially because of this.

-General purpose media are designed to grow as broad a spectrum of microbes as possible.
- They are usually non-synthetic and contain a mixture of nutrients that could support the growth of pathogens and non-pathogens.
-Examples:nutrient agar and broth, brain-heart infusion, trpticase soy agar (TSA).

Enriched:

-This type of medium contains complex organic substances(blood, serum, hemoglobin) or growth factors. Growth factors are specific vitamins or amino acids which some microbes require to grow. These kind of bacteria are termed fastidious bacteria. An example of an enriched medium is blood agar.
-Blood agar, made by adding sterile sheep, horse or rabbit blood, to a sterile agar base is used to grow fastidious streptococci or other pathogens.

Selective:

-This type of medium contains one or more agents that inhibit the groth of certain microbe or microbes, thereby favoring or selecting another microbe to grow. selective media are very important in primary isolation of a specific type of microorganismfrom samples containing dozens of different species.

Differential:

-This type of medium can grow several types of microbes but brings visible differences in them by variances in media color, colony size, gas or precipitates. These variations come from the type of chemicals these media contain and the ways microbes react to them.

Reducing:

-This type of medium contains a substance that absorbs oxygen or slows oxygen penetration, thereby reducing the mediums availability. It is good for growing anaerobic bacteria

Transport:

-This medium is used to maintain and preserve specimens which cannot be processed right away.

Enumeration:

-This medium does not grow bacteria but holds it for purposes of counting.

Assay:

-This type of medium is used to test antimicrobial drugs

Animal inoculation:

-This involves using a live medium such as a chicken embryo or rat, as some microbes only flourish in living things.

Cultures:

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Pure: The container or medium grows only a single type of microbe. AKA axenic. It can be used to create subcultures


Mixed: The container has two or more identifiable, easily differentiated species of microbes
Contaminated: A formerly pure or mixed culture which grows microbes of uncertain identity (i.e. when the culture was exposed to non-sterile instruments or room air)


Definitions

Monoclonal antibodies: antibodies made in the laboratory and designed to target specific substances called antigens.

Nutrient broth: liquid medium containing beef extract and peptone.

Nutrient agar: solid media containing beef extract, peptone and agar
Agar is a complex polysaccharide isolated from red algae. Solid at room temp liquid at boiling. Bacteria cannot eat it.

Beta–hemolysis: complete red blood cell destruction that leaves a clear area. Streptococcus pyogenes, which causes strep throat, does this.

Alpha-hemolysis: partial RBC destruction, which leaves a greenish area. Several species of Streptococcus, many of which are common in the mouth, do this (and are called the viridans streptococci--viridans meaning "green" and referring to the alpha hemolysis)

Treponema pallidum: cause of syphilis. Dark field microscopy is a quick, easy way to see this spirochete and diagnose the disease.

2 Types of Electron Microscopes

-Transmission electron microscope (TEM) - transmits electrons through the specimen; darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts

-10,000-100,000x; resolution 0.25 nm (electron beam 100,000 times smaller than wavelength of visible light)
-always black and white images (but often artificially colored afterwards)

-Scanning electron microscopes (SEM) - provides detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.

-1000-10,000x; resolution 20 nm
-Striking, 3-dimensional images
Big negative of electron microscopes is the sample must be dead (cannot study movement)

Specimen Preparation

- wet mounts & hanging drop mounts -
allow examination of characteristics of live cells: motility, shape, & arrangement
-A Hanging Drop Mount is the easiest and fastest way to find out if the cell has flagella (if the cell can move)
-fixed mounts are made by drying & heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts

Staining

-simple stains-one dye is used
-differential stains-use a primary stain and a counter-stain to distinguish cell types or parts. examples; Gram stain, acid-fast stain and endospore stain
-special stains-capsule and flagellar stains


DIFFERENTIAL STAINS
GRAM STAIN

• The Gram stain classifies bacteria into gram-positive and gram-negative. The video below describes the steps involved in this stain.
  
• Gram-positive (purple) bacteria are generally more susceptible to penicillin and detergents
• Gram-negative (red or pink) bacteria are more resistant to antibiotics

ACID-FAST STAIN

• Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast (these cells won't lose the stain even after applying alcohol)
• Non-acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counter-stained (with a different color basic stain) to see them
• The acid-fast stain is not easy and tuberculosis infections are often missed, particularly in the early stages. One group is training African pouched rats to sniff out tuberculosis samples. This method has proven to be much faster, much cheaper, and more accurate than traditional methods using the acid-fast stain.
  

These are all my notes from the slides and the chapter that I wrote into a flashcard format. I like to print a page of just the terms, then have all the answers/definitions on a different page so I can memorize the information a little bit easier, while testing myself at the same time. I hope it's not too overwhelming. (It has been for me sometimes when I'm studying. I just remind myself, "How do you eat an elephant?" Just one bite at a time!) --Amber Timothy

Flashcard Notes from Ch. 3 : Tools of the Laboratory: The methods for studying microorganisms
1. The 5 I’s of culturing microbes
2. Inoculation
3. Incubation
4. Isolation
5. Selective media
6. Differential media
7. Two most commonly used media
8. Fannie Hesse
9. Pure culture
10. Mixed culture
11. Contaminated culture
12. Enriched medium
13. The total power of the microscope’s magnification
14. Spread plate technique
15. Loop dilution (or pour plate) technique
16. Streak plate method
17. Simple microscope vs. compound microscope
18. Synthetic vs. nonsynthetic media
19. Reducing medium
20. General-purpose medium
21. Media are classified into what three properties?
22. Selective media vs. differential media
23. Carbohydrate fermentation medium
24. Magnification vs. resolving power
25. 3 types of light microscopes
26. Fluorescence microscopy
27. Confocal microscopy
28. Electron microscopy
29. 2 types of electron microscopes
30. 2 types of specimen preparation
31. Chromophore
32. Cationic dye vs. anionic dye
33. Basic dye vs. adicic dye
34. Positive staining vs. negative staining
35. Simple stain
36. Mordant
37. Differential stain
38. Gram stain
39. Acid-fast stain vs. non-acid-fast stain
40. Endospore staining
41. Capsule staining
42. Flagella staining
43. Assay media
44. Enumeration media
45. Transport media
46. Wet mount preparation
47. Hanging drop slide

Answers
1. Inoculation, Incubation, Isolation, Inspection and Identification
2. The implementation of microbes into a medium.
3. After a container of medium has been inoculated, it’s placed in a temperature-controlled chamber to encourage microbial growth.
4. The separation of microbial cells on solid media to create discrete colonies.
5. Nutrient media designed to favor the growth of certain microbes and to inhibit undesirable competitors.
6. Grows several types of microorganisms but is designed to bring out visible differences among those microorganisms.
7. Nutrient broth – liquid medium containing beef extract and peptone, and Nutrient agar – solid media which is a complex polysaccharide isolated from red algae. Solid at room temp, not digestible for most microbes.
8. She thought of agar as a medium.
9. Contains only one species or type of microorganism. Some microbiologists prefer the term axenic, meaning the culture only contains the living thing being studied. Most frequently used culture for lab study.
10. A container that holds two or more known species of microorganisms.
11. A culture into which unwanted microbes of uncertain identity (contaminants) have been introduced.
12. A nutrient medium supplemented with blood, serum, hemoglobin, or some growth factor to promote the multiplication of fastidious microorganisms.
13. Calculate from multiplying the powers of the ocular and the objective lenses.
14. A small liquid sample is placed on the surface of the medium and spread around by a small “hockey stick.” Here, cells are spread over separate areas so they can form individual colonies.
15. The sample is inoculated into cooled, liquid tubes. The tubes are then poured into sterile Petri dishes to solidify. The end result is that the number of cells per volume is so decreased that cells have ample space to grow into separate colonies. In this method some of the colonies will develop deep in the medium itself and not just on the surface.
16. The sample is spread over the surface of the medium to gradually thin it out and separate the cells spatially. The method of choice for most applications.
17. Simple microscope = one magnifying lens. Compound microscope = two lenses: the ocular lens (or “eyepiece”), which sends the virtual image to the eye, and the objective lens (closest to the specimen), which forms the initial image (called the “real image”).
18. Synthetic = the chemical composition of a medium may be completely chemically defined. Nonsynthetic (or complex) = contains ingredients that are not completely definable. Most media are nonsynthetic.
19. Contains a substance that absorbs oxygen or slows the penetration of oxygen in a medium, thus reducing its availability. Important for growing anaerobic bacteria or for determining the oxygen requirements of isolates.
20. Designed to grow a broad spectrum of microbes that don’t have special growth requirements. As a rule, these media are nonsynthetic (complex) and contain nutrients that could support the growth of many bacteria and fungi.
21. 1 – Physical state (medium’s normal consistency): liquid, semisolid, sold (can be converted to a liquid), and solid (cannot be liquefied). 2 – Chemical composition (types of chemicals medium contains): synthetic (chemically defined) and nonsynthetic (complex, not chemically defined). 3 – Functional type (purpose of medium): general purpose, enriched, selective, differential, reducing, transport, assay, and enumeration
22. Selective: nutrient media designed to favor the growth of certain microbes and to inhibit undesirable competitors. Differential: grows several types of microbes but is designed to bring out visible differences among these microbes.
23. Contains sugars that can be fermented (converted to acids), and a Ph indicator to show the reaction. Basis for identifying bacteria and fungi.
24. Magnification = ability to enlarge objects. Refraction of light rays most accounts for magnification. Resolving power = ability to show detail. Shorter wavelengths will provide better resolution.
25. 1- Bright-field: most widely used, specimen is darker than surrounding field. 2 – Dark-field: brightly illuminated specimens surrounded by dark field. 3 – Phase-contrast: has devices in the microscope that transform subtle changes in light waves passing through the specimen into differences in light intensity. Best for observing intracellular structures.
26. Uses UV light and a filter that protects the viewer’s eye. Cells are stained with fluorescent dyes (fluorochromes), then the cells absorb UV light and emit visible light. This technique allows for rapid diagnosis of infections.
27. Uses fluorochromes and a laser light. The laser scans various depths in a specimen, then a computer produces a 3D image. Allows for 40% greater resolution than other light microscopes; can capture a highly focused view at any level of the cell.
28. Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds. Electron waves are 100,000x shorter than the waves of visible light, giving these microscopes tremendous power to resolve minute structures. Magnification between 5,000x and 1,000,000x. 29. Transmission electron microscopes (TEM): transmit electrons through the specimen. Darker areas = thicker, denser parts. Lighter areas = less dense parts. 10,000-1,000,000x magnification, resolution 25 nm. Scanning electron microscopes (SEM): provide detailed 3-dimensional view. Bombards surface of a whole, metal-coated specimen with electrons while scanning back & forth over it. 1,000-10,000x magnification, resolution 20 nm.
30. 1 – Wet mounts and hanging drop mounts: allow examination of characteristics of live cells (motility, shape, and arrangement). 2 – Fixed mounts: made by drying and heating a film of specimen, called a smear, which is dyed to permit visualization of cells or cell parts.
31. The chemical radical of a dye that’s responsible for its color and reactivity. The color-bearing ion (chromophore) is charged and has an attraction for certain cell parts that are of the opposite charge.
32. Cationic dye: also called a basic dye. Carry a positively charged chromophore and are attracted to negatively charged cell components (nucleic acids and proteins). Bacteria contain large amounts of negatively charged substances, so they stain readily with basic dyes, including methylene blue, crystal violet, and safranin. Anionic dye: also called an acidic dye. Carry a negatively charged chromophore and are attracted to positively charged molecules. Bacteria cells have numerous acidic substances and carry a slightly negative charge on their surface, so they tend to repel acidic dyes.
33. In a basic dye, the choromophore is a cation (positively charged ion). In an acidic dye, the chromophore is an anion (negatively charged ion).
34. Positive staining: surfaces of microbes are negatively charged and attract basic dyes (i.e. the dye sticks to cells and gives them color). Used by most procedures. Negative staining: microbes repel dyes and it stains the background. India ink most commonly used.
35. Type of positive staining technique that uses a single dye to add color to cells so they are easier to see. Simple staining tends to color all cells the same color. 36. A chemical that fixes a dye in or on cells and thereby promotes retention of that dye. (Ex. Gram’s iodine in the Gram stain)
37. A technique that uses two dyes (a primary dye and the counterstain) to distinguish between different cell types or parts (ex. Gram stain, acid-fast stain, and endospore stain).
38. A differential stain for bacteria useful in identification and taxonomy. Classifies bacteria into gram-positive and gram-negative. Gram-positive bacteria tend to be killed by penicillin and detergents. Gram-negative bacteria are more resistant to antibiotics.
39. An important diagnostic stain that differentiates acid-fast bacteria (cells that retain a stain in the presence of acid—turns pink) from non-acid-fast bacteria (cells that lose the stain when rinsed with acid—turns blue). Used to diagnose tuberculosis and leprosy.
40. A technique where a dye is forced by heat into endospores (resistant survival cells).
41. A method of observing the microbial capsule. Because the capsule doesn’t react with most stains, it is often negatively stained with India ink. Useful in identifying pathogens because not all microbes exhibit capsules.
42. A method of revealing flagella, the tiny filaments used by bacteria for locomotion. Flagella staining requires a mordant to make the flagella wide enough to see. 43. Used by technologists to test the effectiveness of antimicrobial drugs.
44. Used by industrial and environmental microbiologists to count the number of organisms in milk, water, food, soil, and other samples.
45. Used to maintain and preserve specimens that have to be held for a period of time before clinical analysis or to sustain delicate species that die rapidly if not held under stable conditions.
46. Consists of a drop or two of the culture placed on a slide and overlaid with a cover glass. Disadvantages: the cover glass can damage larger cells, end the slide is very susceptible to drying and can contaminate the handler’s fingers.
47. A more satisfactory alternative than a wet mount. Made with a special concave (depression) slide, an adhesive, and a coverslip from which a tiny drop of sample is suspended. Provides a truer assessment of the cells.

Questions

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1. Which of the following is not one of the "I"s?
a. Inspection
b. Identification
c. Induction
d. Incubation
e. Inoculation

2. A mixed culture is_____________.
a. the same as a contaminated culture
b. one that has been adequately stirred
c. one that contains two or more known species
d. a pond sample containing algae and protozoa

3. Who said, "All life comes from life"?
a. Fannie Hesse
b. Louis Pasteur
c. Antonie Van Leeuwenhoek
d. Robert Koch

4. The term culture refers to the _______ growth of the microorganisms in ________.
a. rapid, an incubator
b. macroscopic, media
c. microscopic, the body
d. artificial, colonies

5. Agar is superior to gelatin as a solidifying agent because agar
a. does not melt at room temperature
b. solidifies at 75 degrees C
c. is not usually decomposed by microorganisms
d. both a and c

6. A subculture is a
a. colony growing beneath the media surface
b. culture made from a contaminant
c. culture made in an embryo
d. culture made from an isolated colony

7.What kind of bacteria tend to be killed by penicillin and detergents?
a. gram-negative bacteria
b. gram-positive bacteria
c. Mycobacterium tuberculosis
d. Bacillus

8. Which type of stain would you use to diagnose tuberculosis or leprosy?
a. Gram staining
b. Special staining
c. Endospore staining
d. Acid-Fast stain

9. What best describes complete RBC destruction?
a. Alpha hemolysis
b. Beta hemolysis
c. Selective hemolysis
d. Differential hemolysis

11. Which bacteria contain large amounts of negative charged substances that stain readily in the crystal violet, methylene blue, and safranin?
a. Gram negative bacteria
b. Gram positive bacteria

12. Bacteria that require growth factors and complex nutrients are termed:
a. enriched media
b. functional type
c. culture
d. fastidious

13. ________ __________ , inhibit most gram-positive bacteria while permitting many gram-negative rods to grow is called:
a. bile salts
b. urea
c. saliva
d. semen

14. A microscope that has a toatal magnification of 1500x when using the oil immersion objective has an ocular of what power?
a. 150x
b.1.5x
c. 15x
d. 30x


Comprehension Questions from Chapter Three

1. What type of media is a complex polysaccharide isolated from red algae?
2. In which type of inoculation technique do some of the colonies develop deep in the medium itself and not just on the surface?
3. Which type of inoculation method is most widely used in the lab?
4. What type of media is important for determining the oxygen requirements of isolates?
5. What type of meda contains sugars that can be fermented (i.e. converted to acids)?
6. What type of media is the basis for identifying bacteria and fungi?
7. Which type of microscope allows for rapid diagnosis of infection?
8. What type of microscope uses special light waves that are 100,000x shorter than the waves of visible light, giving it tremendous power to resolve minute structures?
9. What is the charge that a cationic dye is attracted to? An anionic dye?
10. Do bacteria stain more readily with basic or acidic dyes, and why?
11. What charge does the chromophore have in a basic dye? In an acidic dye?
12. What type of staining technique usually uses India ink?
13. What type of staining technique uses a single dye?
14. What kind of bacteria tend to be killed by penicillin and detergents?
15. What kind of stain is used to diagnose tuberculosis and leprosy?
16. What kind of stain is useful in identifying pathogens?
17. What type of media is used to test the effectiveness of antimicrobial drugs?
18. What type of media is used to maintain and preserve specimens that won't be clinically analyzed right away?
19. What kind of microscope can magnify up to 1,000,000x?
20. Do shorter or longer wavelengths provide better resolution?
21.What are the 5 I's?
22. Who was the person who really thought of using agar in biological experiments?
23. What type of media contains pure organic and inorganic compounds in an exact chemical formula?
Comprehension Answers from Chapter Three

1. Nutrient agar
2. Loop dilution (or pour plate) technique
3. Streak plate method
4. Reducing media
5. Carbohydrate fermentation media
6. Carbohydrate fermentation media
7. Fluorescent microscope
8. Electron microscope
9. A cationic dye is attracted to negatively-charged molecules. An anionic dye is attracted to positively-charged molecules.
10. Bacteria cells stain readily with basic dyes (which are attracted to negatively charged cell components) because bacteria contain large amounts of negatively charged substances.
11. In a basic (also called cationic) dye, the chromophore is a cation (positively charged ion). In an acidi (also called anionic) dye, the chromophore is an anion (negatively charged ion).
12. Negative staining
13. Simple staining
14. Gram-positive bacteria
15. Acid-fast stain
16. Capsule staining
17. Assay media
18. Transport media
19. Electron microscope
20. Shorter wavelengths provide better resolution.
21. Innoculation, Isolation, Incubation, Inspection and Identification
22. Fannie Hess
23.Synthetic

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