Stain Uses– This is used to detect presence of bacteria and morphology of bacteria.
Gram Stain Primary Stain
- First stain used in the Gram
- This increases the binding
- of the primary dye in the Gram stain.
Acetone Alcohol –
is used in the gram stain.
is used in the Gram stain
Counter Stain:(Secondary Stain)
Comments Special Steps
- Uses:–This stain detects spores.
- Malachite Green- This is used in the spore
- stain, it stains the spores green.
- No mordant is used in the spore stain.
- Water –is used as the decolorizing
- agent in the spore stain.
- Safranin –is used in the spore stain.
- In the spore stain procedure
- -heat fix the cells to the slide
- -steam primary stain
- (Malachite Green) for 10-15 minutes to drive the stain into the spore.
- - Any spore outside of a cell is a free spore.
- - Cells will stain red.
Acid Fast Stain
Counter StainSecondary Stain
Comments and Special Steps
- Stain Uses– This stain detects acid-fast
- cells, such as Mycobacterium tuberculosis
- Carbol Fusion – is the primary used in the
- acid fast stain. The primary stain is steamed
- for 10 minutes (in the ZN method)
- There is no mordant used in the acid fast stain.
- Acid Alcohol – This removes stain from the
- non-acid fast cells.
- Methylene Blue –is used as the counter stain
- in the Ziehl-Neelsen (ZN) acid fast stain.
- Brilliant Green – is used as the counter stain
- in the Kinyoun acid fast stain
- - Steam primary stain for 10 minutes (ZN)
- - The decolorizing step is sensitive.
- - The waxy cell wall makes acid-fast cells difficult to stain; once stained, they are difficult to destain
Nigrosin Negative Stain
somments and special steps
- Determines morphology and cellular arrangement in bacteria that are too delicate to withstand heat fixing
- Nigrosin –is used in the negative stain.
- no mordant
- no decolorizing agent
- no counter stain
- Do not heat fix in the negative stain.
Anthony’s Capsule Stain
Counter Stain(Secondary Stain)
Comments and Special Steps
- Used in Identifying Capsules.
- Anthony’s Crystal Violet–is used in the capsule stain.
- Copper Sulfate–Decolorizes the capsule (but not the cell.)
- There is no counter stain in the capsule stain.
- -Do not heat fix the cells in the capsule stain.-Litmus milk protein is what accounts for the background staining.Cell and background stain purple; capsule clear
This is the use of specific methods and sterile materials to exclude contaminating microorganisms from an environment.•
This is a device employing steam under pressure used for sterilizing materials that are stable to heat and moisture. (15 psi, 121 C for 15 minutes.)
Liquid media used to grow bacteria.
Visible mass of bacterial cells arising from a single cell.
The process of removing or destroying all microorganisms and viruses on or in a product.•
Transfer Loop (Inoculating Loop)–
Thin wire with a loop on the end used in transferring bacteria to a culture medium.
Inoculating Needle (Transfer Needle)–
Thin straight wire used for stabbing agar (to get culture away from oxygen), or for fine manipulations.
Population of organisms descending from more than one kind of cell.
These are colonies separated from other colonies on a petri dish.
This is a population of organisms descending from a single cell.
The simplest and most commonly used technique for isolating bacteria, a series of successive streak patterns is used to sequentially dilute an inoculum on the surface of an agar plate (non-quantitative dilution.)
Mix culture in a drop of water to give a smooth, even suspension (used for making smears from solid media; i.e. plate or slant)
This technique helps to retain the cells on the slide throughout the staining process.
This is a staining technique that employs one basic dye to impart color to cells.
This is the technique in which the cell is stained but the background remains unstained. Examples: Gram Stain, Spore Stain....
Staining technique in which the background is stained and the structure of interest is not stained. This staining technique may employ an acidic dye to stain the background against which colorless cells can be seen (Example: Nigrosin negative stain.) In the Anthony’s Capsule stain, the basic dye Crystal Violet is used to stain the background and the cell allowing the clear capsule to be observed.•
This is a type of staining procedure used to distinguish one group of bacteria from another by taking advantage of the fact that certain bacteria have distinctly different chemical structures in some of their components.
This is the first dye applied in a multi –step differential staining procedure; generally stains all cells.
This is the substance that increases the affinity of cellular components for a dye.
Decolorizer (Decolorizing Agent)–
Selectively removes the primary dye from one type of cell or cell structure.
The stain applied to impart a contrasting color to bacteria that do not retain the primary stain.
This is a differential stain used to detect the presence and location of spores in bacterial cells.
This is used to distinguish between Gram positive and Gram negative cells. This is probably the most important and widely used microbiological differential stain.
A typical, actively growing cell
This is a kind of resting cell, characteristic of a limited number of bacterial species (Bacillus, Clostridium), highly resistant to heat, radiation, and disinfectants.
This is in bacteria, a complex, highly ordered sequence of morphological changes during which a bacterial vegetative cell produces a specialized cell greatly resistant to environmental adversity.
This is the sum total of the biochemical and morphological changes that an endospore or other resting cell undergoes before becoming a vegetative cell.
This is located at the end of the cell.
This is located in the middle of the cell
This is when a spore is free of the mother cell after degeneration.
Lowenstein –Jensen Medium
This is used for the isolation of mycobacterium. Egg supplement enhances the growth of Mycobacteriumand malachite green prevents growth of the majority of contaminants.
This is a waxy substance found in cell walls of acid-fast bacteria.
This is a Granuloma formed in tuberculosis.
This is an undefined medium consisting of skim milk and azolitman. Skim milk provides nutrients for growth with lactose as the carbohydrate source and casein as the primary protein source.
This is culture medium that inhibits the growth of certain microorganisms and therefore favors the growth of desired microorganisms.
This is culture media that allows visual identification of certain types of microorganisms.
This is a medium in which the chemical composition and quantity of every component is known.
This medium is for growing bacteria that has some ingredients of unknown chemical composition.
All Purpose Growth Media
Complex media that is routinely used to culture bacteria. Usually inexpensive and easy to prepare (Example:TSA).
This media is used to isolate organisms which:•1.Can or must use compounds such as a carbon source or vitamins which have been added to the media. Example blood agar.•2.Can grow without compounds which have been omitted from the media; usually a synthetic media. Example citrate media
Antibiotic Sensitivity (Kirby –Bauer Method)–
This is testing used to determine the susceptibility of bacteria to various antibiotics. This is typically used to measure the effectiveness of a variety of antibiotics on specific organisms in order to prescribe the most suitable antibiotic therapy.
Why do you avoid turning the petri dish lid upside down when you remove the lid from the petri dish?
This will keep other microbes from contaminating the media.
Why do you incubate the plate inverted?
This keeps water from running onto the culture and contaminating our culture.
What is the Gram reaction of dead cells?
The cells will appear to be gram negative because the degrading cell wall cannot retain the primary stain and it will appear to be red.
Is the endospore stain a differential stain? If so what is the decolorizing agent?
A. Yes.•B. Water is used as the decolorizing agent.
Is the acid fast stain a differential stain? If so what is the decolorizing agent?
A. Yes.•B. Acetone Alcohol
Is the capsule stain a differential stain? If so what is the decolorizing agent?
•A. Yes.•B. Copper Sulfate.
What are the two most obvious visual observations that indicate that you do not have a pure culture?
•A.The culture looks different than the other organisms. (Different Morphology.)•B. The Culture will not be on the streak line.
On an isolation streak plate, what might account for differences in colony size of a pure culture?
•A. Availability of nutrients.•B. Time of incubation.
In an acid-fast stian, what color are non –acid fast organisms?
•A. Blue (Ziehl-Neelsen) or green (Kenyon)
The acid fast stain is important in identifying pathogenic microorganisms, name 2 acid fast pathogens and briefly describe them and the diseases they cause?
•A. Tuberculosis–(Mycobacterium tuberculosis) Two manifestation of this disease include primary and secondary. Primary stage in a healthy person causes mild flu like symptoms. Necrotic lung inflammation occurs in the secondary stage; secondary is usually associated with a weakened immune system.•B. Mycobacterium leprae–Causes Leprosy, characterized by granulomas on face & extremities; loss of nerve function.
What role do capsules play in the disease process?
•A. Capsules protect bacteria from phagocytosis.
Are there any water wash steps in the Anthony’s capsule stain? Explain?
•A. No.•B. This is because the stain is water soluble, and because some slime layers/capsules are water soluble.
Why do you report sensitivity to an antibiotic instead of just the size of the zone of inhibition?
•Migration through the agar is dependent on chemical characteristics (molecular weight; hydrophobic/hydrophilic nature) and must be related to clinical effectiveness.
14. How do the following factors influence the size of the zone of inhibition?
•A. Hydrophobic ( lipid soluble ) Antibiotic (dislikes water)–•This antibiotic will produce a smaller zone of inhibition.•B. Hydrophilic ( water soluble ) Antibiotic (Water loving.–•This antibiotic will diffuse easily in the agar medium and will give a large zone of inhibition. •C. Small molecular weight –•Antibiotics will diffuse further in a small molecular weight than a large molecular weight, producing a larger zone of inhibition.•D. Large molecular weight –•Antibiotic will diffuse less than a small molecular weight, producing a small zone of inhibition.
Anaerobic Culture Jar
- •Tests for:•A. Ability of organisms to grow in the absence of oxygen•(Note: Ability to grow in presence of oxygen has been established.)•Test Results:•A. Growth under anaerobic conditions.•1. Obligate Aerobe: -No growth (Catalase Positive +).•2. Facultative Anaerobic: -Growth (Catalase +).•3. Aerotolerant: -Growth (Catalase -).
- •The white methylene blue strip and the open packet which has discharged H2 and CO2 gases.•The palladium, contained in the packet, catalyzes the conversion of H2 and O2 to water.
MacConkey –E. coli
Pink/red indicates acid production from lactose fermentation.•A. MacConkey agar is used to isolate and differentiate members of the Enterobacteriaceae –(Growth indicates the bacteria is most likely Gram negative.)•1. E. coli–Purplish red color; large halo around growth &/or on streak indicates bacteria ferments lactose
Mannitol Salt Agar (MSA)
A. This synthetic (defined) agar is both selective and differential. It favors organisms capable of tolerating high sodium chloride concentration and also distinguishes bacteria based on their ability to ferment mannitol. It is used to differentiate pathogenic Staphylococcusspecies, which ferment mannitol, from the less pathogenic members of the genus Staphylococcusand from Micrococcus.•No Growth indicates the organism is most likely Gram negative.
- DNase agar is a differential medium that tests the ability of an organism to produce an exoenzyme, called deoxyribonuclease or DNase, which hydrolyzes DNA. DNase agar contains nutrients for bacteria growth and DNA. DNase activity can be detected by covering the media with HCl after incubation which precipitates the intact polymerized DNA, leaving a clear halo around the bacteria that produce DNase.NOTE: Some DNase agars contain cationic dyes, such as methyl green and toluidine blue, that form a colored complex with the negatively-charged DNA. A clear halo around the streak after growth indicates DNase activity. However, these dyes may be non-specifically inhibitory, especially to Gram positive bacteria.
- Clearing around the streak after addition of HCl indicates a positive DNase test.
E. colion EMB (Eosin Methylene Blue) plate
•A. This is a selective and differential medium used for isolation and differentiation among members of the Enterobacteriaceae the enteric (gut) bacteria. Enteric bacterium are facultative anaerobic Gram negative rods.•B. The difference in color is due to the degree•of acid production.•1. E. coliproduces a metallic green sheen.
- •1. Gamma Hemolysis –Grows on the media but does not hemolize blood. (Staphylococcus epidermidis)•2. Beta Hemolysis–Clear area extending from the streak line; lyses blood cells and completely degrades hemoglobin. (Streptococcus pyogenes)•3. Alpha Hemolysis–Partial clearing with greenish cast. (Streptococcus sanguis; Streptococcus bovis)
- •A. Blood agar is used for the cultivation of fastidious organisms requiring specific growth factors not available in other media.•The degree to which these exotoxins hemolyze erythrocytes is a useful diagnostic tool in the identification of the genera Streptococcus, Aerococcus, and Enterococcus.•
- •Beta Hemolytic –Complete clearing around colonies.
- •C.Alpha Hemolysis–•Clearing with greenish cast. Red blood cells are lysed but hemoglobin is not completely degraded.
- •D.Gamma Hemolysis–•Bacteria can grow on medium but it does not lyse the blood cells.
Lowenstein Jensen Slantis used to grow acid-fast bacteria
- •1. Mycobacterium smegmantis(similar to M. tuberculosis) –Growth appears to be a bread crumb appearance with a rough-looking texture which is a kind of a yellowish brown color.•2. Know 2 diseases caused by acid fast bacteria
- For the cultivation and isolation of Mycobacterium. LJ is a glycerated egg-potato medium. Malachite green dye is added as a partial inhibitor to microorganisms other than mycobacteria. Ribonucleic acid increases the percentage of positive cultures. Mycobacteria have a “dry bread-crumb” appearance.
Litmus Milk Medium
Tests for:1. Acid (A) –Pink color.2. Alkaline (K) –Blue color.3. Reduction of Litmus (R) –White Color.4. Proteolysis/Peptonization (P)Peptonization –Clearing [Brown straw color or dark blue (Alkaline)]5. Curd (C) –Curd (solid); often with acid.6. Rennin Curd (RC) –curd with lavender top
Sugar fermentation Tube
•Ability to ferment sugar:•A. Acid production (A) –Media green / yellow (positive test)•B. Gas Production (G) –Bubbles in Durham tube (Positive test)•C. No acid or gas production –No color change, Purple:•( -) negative test.•Note:•Deamination turns the media alkaline (K); reported as (-) negative fermentation test.Purple glucose broth.
Bile Esculin Agar
•Tests for:•A. Ability to hydrolyze esculin in the presence of bile.•Test Results:•A. More than ½ of the tube turns black or dark brown in 72 hours or less.
Nitrate Reduction TestStab buttStreak slant(we have agar, not broth)•
Testing for:•A. Reduction of nitrate, NO3 to nitrite, NO2 (Which is detected by reagents)•Phase 2:•A. Add 5 drops sulfanilic acid, then 5 drops a –naphthylamine.•Phase 2 results:•A. Red color indicates nitrite, NO2 is present –Positive Test ( NO3 was reduced ).•B. Other compounds ( not detected by reagents)•Phase 3:•B. Add a “Pinch” of powdered zinc.•Phase 3 results:•B. If NO3, was not used, it will be reduced to NO2 by zinc: -Red / Pink color is a negative test.•-Clear reagents indicate a positive test.
Sulfur ReductionStab Butt
•Testing for:•A. Sulfur reduction; formation of H2S which is detected by the black iron (ferrous) sulfide precipitate.•Test Results:•A. Positive H2S test –Black precipitate in butt and or on surface of media.
•Testing for:•A. The enzyme tryptophanase, which converts tryptophan to indole; detected with Kovac’s reagent•Test Results:•A. Red color in alcohol (top) layer.
Methyl Red (MR) Test(IMViC)
•Testing For:•A. Glucose fermentation to produce large amounts of stable acids.•B. Divide MR/VP broth before adding MR reagent.•Test Results:•A. Red –Positive for stable acid production.•B. Yellow –Negative for stable acids.•C. Orange -Inconclusive.
Vogues –Proskauer (VP) Test(IMViC)
•Test for:•A. Glucose fermentation to produce 2, 3 –butanediol and acetoin.•B. Divide MR / VP broth before adding VP (Barrett's) reagents.•Test Results:•A. Media Should turn milky white after adding VP A then clear after adding VP B; cap and carefully mix well•once a minute for about 5 –10 minutes.•1. Positive –Red Color.•2. Negative –Brown or Copper Color.
Simmons citrate AgarStreak Slant with light inoculums(IMViC)
•Tests for:•A. Ability to grow on citrate as sole carbon source (CO2 produced from citrate.)•Test Results:•A. Growth –Slant appears to be blue.•B. If slant is green with growth –incubate additional 24 hours or redo.•C. No Growth –Slant appears to be green.•Positive Negative Uninoculated•Test Test Test
•Testing for:•A. Ability of organism to hydrolyze starch.•Test Results:•A. Cleared area around streak.
Test ResultsPositive Test (+):all pink at 24 hr Weak Positive (wk+):partially pink by 24 hr to 6 days Negative Test (-):orange or yellow at 6 days
•Testing for:•A. The respiratory enzyme, cytochrome oxidase.•B. Transfer culture to sterile Q-tip, add 1 drop oxidase reagent•Test Results:•A. Colorless Tetramethyl –p –phenylenediamine red. (TPPD red) Yields TPPD ox deep purple / blue.•1. Positive Test (+) –Blue color in 10 –15 seconds for G-or G+.•2. Due to the thicker cell wall, blue color in 15 –60 seconds that continues to develop is Positive Test (+) for G+.•3. Negative (-) –No color, yellow, brown; or blue that takes longer than 60 seconds to develop and/or fades quickly.
•Testing for:•A. The enzyme catalase, which converts H202 to H20 and O2 (bubbles).•Test results:•A. Bubbling is a positive test; H2O2 is unstable so run a positive control if test is negativePositive Test. Negative Test.
Gelatin LiquefactionStab Butt.
•Tests for:•A. Ability to hydrolyze (liquify) gelatin.•Test Results:•A. Cool before reading test (gelatin melts above 28 degrees Celsius);•B. Partial or complete liquification is a positive test.•C. If solid, test is negative.