Microbiology Lab Practicum #1
o Rhizopus stolonifer
(cause of Black Bread Mold)
Asperigillus
(causes aspergillosis in the lungs) Aspergillus species are commonly found in soil on plants. Roughly 180 species have been indentified. They are charactorized by green to yello or brown gradular colonies with white edge. One species A niger, produces distinctive black colonies. Vegetative hypae are hyaline (unpigmented) and septate. The aspergillus fruting body is distinctive, with chains of conidia arising from one (uniseriate) or two (biseriate) rows of phialides attached to a swollen vesicle at the end of an unbranched conidiophore. the conidiophore grows from a foot cell in the vegatative hypha. fruting body structure and size, and conidia color are useful in species were incapable of sexual reproduction, but evidence is mounting that under the right enviroment conditions viable ascopores can be produced. A fumigatus and other species are opportunistic pathogens that cause aspergillosis, and umbrella term covering many diseases. Immunocapromised patients are at higher risk of infection. Invasive aspergillosis is most serveer form and has a high mortality. It results in necrotizing pneumonia and may spread to other ograns such as the heart or central nervous system. antifungal medication can be used in treatment but identification of the pathogen to species level is necessary because of their different sensitivities. Allergic aspergllos may occure in individuals who are in frequent contact with the spores and become sensitive to them. Subsequent contact produces symptoms similar to asthma. aspergilloma involves colonization of the paranasal sinuses or lings, resulting in abscess formation. It is frequently asymtomatic and resolves without treatment, but in serious cases can be fatal.
Microscopy
Field of view: the total area that can be seen at a given magnification. Parfocal: the ability for a microscope to remain focused when switching between objects. Index of refraction (n): the degree to which light bends as it pass through median, Abbe's equation: D=0.G IX/)Sin D- resolution or ability to distinguish two seperate points. -/_ wavelenght of light (nm) N- Index of refraction O= half of the angel of the cone of light.
Acid Fast Staines
The acid fast stain is a differential stain used to detect cells capable of retaining a primary stain when treated with acid alcohol. It is an important differential stain used to identify bacteria in the genus Mycobacterium, some of which are pathogens (M. Leprae and M. Tuberculosis. Carbolfushion.
Two stains often used in negative staining are
congo red and eosin stain
The Negative Stain
purpose: to stain cells and visualize fraigel heat senstive cells and observe cell size, morphology. Materials: - T.T. rack - tooth pick - Congo Red dye - your mouth. 1. place 1/2 loop of congo red on edge of slide 2. harvest bacteria onto congo red. 4. use a cheap new slide as a tool to smear. 5. air dry and store in chest.
mycology
the study fungi, non-photosynthetic, cell wall made of chitin and cellulose. Aborsbative hetertrophs, excreates digestive enzymes outside of the cell.
Microscopy
view colored threads, blood smears and letter e. Is used to identify micro-organisms. D= Lambda/NA condenser + NA objective . N= index of refraction. Parfocal= ability to stay in focus. Is used to identify micro-organisms.
Sabourandi dextrose agar
which has a PH of 5.6, is used to isolate fungi that outgrow most bacteria at this PH.
Acid Fast Stain
(causes tuberculosis in lungs) Mycobacterium tuberculosis (causes tuberculosis in lungs)The presence of mycolic acid in the cell wall of acid fast cells a higher affinity for the primary stain and resistance to decolorization by an acid alcohol solution. A variety of acid fast-staining procedures are employed, two of which are Ziehl-Neelsen (ZN) method and the Kinyoun (K) method. These differ primarily in the ZN method uses heat as part of the staining process (this is in addtion to heat fixing the preparation) whearas the K method is a "cold" stain (which is still heat fixed). In both protocols the bacterial smear may be prepared in a drop of serum to help the "slippery" acid fast cells (they are waxy after all) adhere to the slide. The two methods provide comparable results and the choice of method comes down to lab conventions and personal preference.
Candida Albicans
(causes yeast infections) Candida Albicans is part of the normal respritory, gastrointestinal and female urogenital tract floras, but it is also the cmost common fungal opportunistic pathogen. It is the causative agent of thrush in the oral cavity, vulvovaginitis of the female genitals and cutaneous candidiasis of the skin. Systemic candidiasis may follow infection of the lungs, bronchi or kidneys. Entry into the blood may result in endocarditis. The ability to attach epithelial surfaces of mucous membranes and sunsequently penetrate to deeper tissues are important virulence factors. individuals most susceptible to Candida infection are diabetic, those with immunodeficiency, catheterized patients, and indivvidual taking antimicrobial medications. Treatments very depending on the location of infection and the state of patient. Candida albicans cells are round oval that reproduce by budding and are 3 um-6um in diameter. Sometimes the cells seperate after budding, but other times they remain attached, forming septate chains of two types: pseudohyphae, which are narrower at their junction and ture hyphae, which are not. Both types branch and produce aseual blastospores. The ability to switch growth patterns between pseudohyphae and true hyphae may increase virulence. Large, round thich walled chlamydospores form at the end of pesudohypae. The sexual life cycle is similar to S cerevisia.
Penicillium
(penicillin antibiotic, cheese, lung disease, etc.) Members of the Penicillium are ubiquitous, being found in the air, soil. and decaying organic matter worldwide. Over 200 species have been identified. They produce distinctive green, powdery, radically furrowed colonies with a white apron and light colored reverse surface. The hyphae are septate and thin. Distinctive Penicillium fruting bodies, consisting of metulae, phialides, and chains of spherical conidia, are located at the ends of branched or unbranched conidiosphores. Although not an improtant feature in laboratory identification, sexual reproduction occurs under proper conditions and results in formation of ascospores with in an ascus. Penicillium is best known for its production of the antibiotic penicillun but it is also a common contaminant.
Staphylococcus aureus
(skin infection) Staphylococcus aureus causes a wide range of human infections from pimples and boils to pneumonia, food poisoning and surgical wound infections, and it is a significant cause of hospital associated infections. After penicillin's initial success in treating S aureus infection. penicillin-resistant S, Aureus became a major threat in hospitals in the 1950's, requiring the use of methicillin. In the 1980' methicillin-resistant S aureus (VRSA) in a patient in the united states was reported.
Conditions
1. Bacteria is present 2. Bacteria is present fermentation of Sugar.
Candid Albicans
1. Found in respritory, gastro intestinal track, female urogential track. 2. predisposing factors: ph, hormones levels, temperature, salinity. 3. Leads to thrush abnormal growth in cavity, yeast vulorvaginitis, endocardioitis, systemic infections.
List 3 reason for heat-fixing bacterial smears.
1. Kills bacteria 2. Makes them adhere to slide 3. Coagulates cytoplasmic proteins to make them more visible.
Simple Stain stain the __________ microorganisms and not the ________ background; negative stains stain the _________background and not the cell.
1. Microorganism 2. Background 3. Background 4. Cell
What are two types of asexual spores that exist for filamentous fungus?
1. Mycobacterim 2. Streptomyces.
Preparing the Bacterial Smears
1. Place a small drop of H2O to clean slide using a steril loop. - frosty side up -lable-name/intials - "gram stain" - "CF: SE" Why h2o? Acts as an Adhesive for Bacterial transfer. 2. Using palm-up (pencil technique) stir in bacterial transfer. - Stir in CF SE one-by-one into same drop of H20. Sterilize needle each time. -If drop dries before innoculation, start over! -when done place bacteria-half screw caps on refrigdge. 2. Air dry-all the way---> slide warmer 4. Heat fix (3 pass technique) using close pin. Heat Fix purpose: to stick specimen on slide/adhere cells to slide. Kill specimen. Mechanism: Protein denatures at high temperature. Coagulates. * Bacterial 'carcus' remains. Distingushing different bacteria using Gram Stain by Hans Christian Gram (1884) Purpose: to stain bacteria and differentiate between two different cell walls types and structure, morphology and arrangement. Why use? Correct Antibiotics. -You can get inaccurate results under exposure. -over drying the slide all day. -If broth was sucrose, bacteria using it and stiring as starch.
A spirochete that is usually stained by a negative stain is __________________, This organism causes the disease __________. Another spirochete is ______________, which causes the disease _______________.
1. Tremponema 2. Syphilis 3. Borrelia 4. Lyme Disease.
Mold Multicellular
1. procreases via binary fission. 2. Contains hyphae or individual filaments and looks cottonish. 3. produces "spores" (asexual & sexual)
Fungi- Describe some general characteristic of fungi
1. vegetative structures 2. molds and fleshing fungi: the thallus (body) of a mold or fleshy fungus consists of long filaments of cells joined together these filaments are called hypha. B. Septa-cross walls C. Speta hyphae- cross wall divide into D. eoenocytic hypae- no speta and appear long E. mycelium-conditional enviroments from filamentous mass myceli.
Two Divisions
1. yeast (unicellular), procreate via budding, pseudohype (chain of daughter cells) 2. mold
Differential Stains allow a microbiologist to:
Allows microbiologist to detect differences between organisms or differences between parts of the same organism.
Streak Plate
A microbial culture of two or more species is said to be a mixed culture, whereas pure colonies contain only a single species. Obtaining isolation of individual species from a mixed sample is generally the first step in identifying an orgranism. A commonly used isolated technique is thestreak plate. In the streak plate method of isolation, a bateria smaple (always assume to be a mixed clture) is streaking over the surfect of a plate of agar medium. During streaking, the cell density decreases, eventually leading to individual cells being deposited separtely on the agar surface. Cells that have been isolated been sufficently isolated will grow into colonies consiting only of the original cell type. Because some colonies form from individual cells and other pairs . chains, or clusters of cells,, the term colony forming unit is more correct description of the original colony. Several patterns are used in streaking an agar plate, the choice depends on the source of inoculum and microbiologist's preference. Although streaking patterns range from simple to more complex, all are designed to separate deposit cells (CFU) on the agar surface so individual cells (CFUs) grow into isolated colonies. A quadrant strak or a T-streak is generally used with samples suspected of high cell density, whereas a simple zigzag (continous streak) pattern may be used for samples containing lower cell densities.
Besides the gram stain, other differential stains test for the presence of
Acid Fastness, Capsules, Spores, Flagella.
The decolorizing solution we use in the gram stain is ______ .
Alcohol/Acetone
In good gram negative stains, what should background of the slide look like?
Bacteria cell is unstained against a dark background because the cells are made visible against a dark background. Distortions of the cell size and shape are minial because heat fixing is not necessary and the cells do not pick up any of the stain.
Onpg
Bacteria that ferment lcatose typically produce teo enzymes B-galactoside permase, a membrane bound transport enzyme b galactoside an intracellular enzyme that hydrolyzes the disaccharide into B-glucose and b-galactose. Bacteria possessing both enzymes are active b-lacose fermenters. Bacteria that cannot produce B-galactosidase permease cannot ferment B-lactose. Bacteria possess bglactosiase but no (or small amounts of) b-galactoise permease are slow to ferment lactose because it does not enter the cell. P Vulgaras.
Why are most bacteria most easily stained by basic dyes?
Basic stains (where the auxochrome becomes positively charged as a result of picking up a hydrogen ion or losing a hydroxide ion) are attached to the negative charge on the surface of most bacteria.
Why is it best to use cultures that are 24 hours old or less for gram staining?
Because some gram positive loose there ability to retain Crystal Violet complex in as little as 24 hours.
Postive Vs Negative Results
Broth---> not cloudy---->no bacteria---->inconclusive Cloudy--->and------Pink----->bacteria!!! No fermentation, (-) Cloudy---> and----- Red----->bacteria!! (-) No fermentation Cloudy----> and -----> yellow-----> Bacteria !! Fermentation (+)
Caosule Stain
Caosules are composed of mucoid polysaccharides or polypepties thaat repel most stains because of their nuetral charge. The capsule stain technique takes advantage of this charactoristic by staining around the cells. Typically, and acidic stain such as congo red or nigrosion, which staings the background and basic stain that colorizes the cell proper are used in combination. The capsule remains unstained and appreas as a white halo between the cells and the colored background. This technique begins as a negative stain; cells are spread in a film with an acidic stain and are not heat fixed, heat fixing causes the cells to shrink, leaving and artifical white halo around them that may be emulsified in a drop of serum to promote their adhering to the glass slide.
Capsule Stains
Capsules are composed of mucoid polysaccharides or polypeptides that repel most stains because of their of their neutral charge. The capsule stain technique take advantage of the characteristic by staining around the cells. Typically, an acidic stain such as congo red or nirosin, which stains the background and a basic stain that colorizes the cell proper are used in combination. The capsule remains unstained and appreas as a white halo between the cells and colored background. The technique begins with an acidic stain and are not heat fixed . Heat fixing causes the cells to chrink, leaving an artifactual white halo around them that might be interpreted as a drop serum to promote their adhering to the glass slide. The capsule stains a differential stain used to detect cells capable of producing an extracellular capsule. Capsule production increases an extra capsule. Capsule production increases virulence in some microbes (such as anthrax Bacillus Anthracis and the pneumococcus streptocpccus pneumoniae) by making them less vulnerable to phagocytoisis.
What is a chromophore? An auxochrome? What kind of charge does the auxochrome have in basic stains.
Chromophore- the portion of the chromogen that gives it color. Chromogen- colored molecule. Chromogen have multiple chromophores which add intensity to color. Auxochrom-is the charge portion of a chromogen and allows it to act as a dye through ionic or covalent bonds between chromogen and the cell.
What do gram negative cells look like after the decolorization step in the gram stain? Why?
Clear because all of the color has been removed and the gram negative cell wall have a high lipid content (because of the outer membraine and a thinner peptidoglycan layer than in gram positive cell walls. The Alchohol/Acetone in the decolorizer extracts the lipid making the gram negative cell wall more porous and incapable of retaining Crystal Violet Iodine Complex there by decolorizing it.
Why do you need a counterstain in the gram stain?
Counter Stain allows gram negative cells to be colorized.
In the last step in the gram stain, gram negative cells are colored by safranin, which is a _______.
Counterstain.
The Primary Stain the Gram Stain is ________. What do the cells look like after application of this primary stain?
Crystal Violet, Purple.
In the gram stain a _______ step occurs between the application of 2 _____ stains.
Decolorization, Basic.
The most critical step in the gram stain is _______ step.
Decolorization.
Endospore Stain
Done to detect the presence and shape of endospores in bacteria. Bacillus Cereus, Bacillus Coagulans, Bacillus Magaterium, Bacillus Subtilis.
What segment of the human population is most susceptable to infection by fungi
Gardens and Farmers
What do the gram negative cells look like on successful completion of the gram stain? Gram Positive cells?
Gram Negative cells are red. Gram Positive are purple.
Name 3 gram negative bacterial genera and 2 gram positive bacterial genera other than the genera used in the exp.
Gram Positive: Bacillus and Stapphylococuss
The most commonly used differential stain in bacteriology is the_____ stain.
Gram Stain.
Negative stains are used to determine____________and __________.
Morphology and Arrangement.
Yeast and Fungi
Identification mold yeast- largely relies on visual inspection of fungal colonies.
Simple Stain (wet mound of cells)
Materials- -Due (metholyne blue) -Glass Slide with frost. -Chem wipe to clean slide (no microscope) -tooth pick -cover slide Procedure: 1. Place 1 drop of metholyne blue on slide. Do not touch. 2. Harvest your cheek via tooth pick. 3. Stir n cheek cells. 4. Observe at low power (10x) and high power lens (40x)
Methyl Red and Voges-Proskauer
Methyl Red and Voges-Proskauer tests are components of IMVic Battery of tests (Indole, Methyl Red, Voges Proskauer and Citrate) used to distinguish between members of the family Enterobacteriaceae and differentiate from other gram negative rods. Process or Fermentation. Escherichia Coli, Enterobacter aerogenes.
Mehyl Red Voges Proskauer test
Methyl red and Voges-Proskauer (MR-VP) broth is a combination of medium used for both methyl re (MR) and Vogels Proskauer (VP) tests. It is simple solution containing peptone, glucose and phospate buffer. The peptone and glucose, and phosphate buffer. The peptone and glucose provide protein (with its nirtogen) and fermentation carbohydrate, respectively and the postasium phosphate resists ph changes in the medium. The MR test is designed to detect organisms capable of performing mixed acid fermentation, which overcomes the phosphate buffer in the medium and lowers the pH. The acids produce by these orgranisms tend to be stable, where acids produce by other orgranisms tend to be unstable and subsequently are converted to more neutral products. Mixed acid fermentation is verified by the addition of methyl red indicator dye following incubation. Methyl red is red indicator dye following incubation. Methy red is red at pH 4.4 and yellow at pH 6.2 Between these two pH values, it is various shades of orange. Red color is the only true indicator of positive results. The Voges-Proskauer test was designed for orgranisms that are able to ferment glucose, but quickly convert their acid products to acetoin and butabediol. Adding VP reagents to medium oxidizes the acetone to diacetly, which turns the reacts gunidine nuclei from peptone to produce a red color. A postive VP test, therefore is red and a ne
Name 3 common basic stains?
Methylene Blue, Crystal Violet, and Safranin.
Iodine acts as a ______ in the gram stain. What is the purpose of adding iodine?
Mordant, and enhances the Crystal Violet staining by forming a Crystal Violet Iodine Complex.
In the medical Laboratory, bacterial characteristics such as morphology and arrangement are usually determined by a ______ stain.
negative.
What are stains? what do they consist of?
Staining simply means coloring the micro-organism with a dye that emphasizes certain structures. (must be heat fixed) 1. Smear- thin film of material bunsen burner a couple times. 2. fixed- to attach the microbes to the slide and kill them.
What will happen to gram positive cells if you add to much alcohol in the gram stain?
Over colorization then by leaving decolorizer on to long and get a redish gram positive cell.
Phenol Red broth
PR broth is used to differentiate members of Enterobacteriacease and to distinguish them from other gram negative rods. Also, is used to distinguish between Gram-positive fermenters, such as streptococcus and lactobacillus species. Alchigenes faecalis, Escherichia Coli, Providencia stuartii, Streptococcus gallolyticus.
Members of the genus Penicilium are well known for producing the antibiotic _________
Penicillin
Phenol Red test
Phenol Red test (PR) broth is a differential test medium prepared as a base to which a carbohydrate is added. Included in the base medium are petone and the PH indicator Phenol Red. Phenol Red appears yellow below PH 6.8, pink magenta above ph 7.4 and red between. During preperation the ph is adjusted to approximately 7.3 so it apprears red. Finally, an inverted durham tube is added to each tube as an indicator of gas production. Acid production from fermentation of carbohydrates lowers the pH below the neutral range of the indicator and turns the medium yellos. Deamination of amino acids supplied by peptone results in all alkaline reaction from the ammonia (NH3) that is produced which raises the PH and turns the broth pink. Gas production also from fermentation is indicated by a bubbl or pocket in the durham tude where the broth has been displaced. (Streptococcus and Lactobacillus).
What do gram positive and gram negative cells look like after the mordant is added in the gram stain?
Purple
Aspectic Technigue
Purpose: to handle bacteria cultures and prevent contatination from occuring. Innoculation_ to introduce microbes into environment. 1. Obtain inoculation loop, lable 2. Flame-sterilized loop. 3. Asecptically harvest cells from source. 4. Make sterile culture (start) of selected bacteria A. Citro Bacteria Frundill (CF) B. Staphylococcus epidermis (SE) 5. loosly put can back on. 6. Incubate in metal can 37.
Isolate of bacteria via streak plate
Purpose: to spread bacteria around an plate and grow sperately isolate colonies. note use: one loop of bacteria for whole experiement. -flame sterile tube ect. - lable agar plates. (name, day, time). Step 1: Streak a loopful into lawn then flame! Then sterlize. -when extracting bacteria tap volvx tube to "wake" them up. Step 2: make 6-8 directional streaks using edge of loop. The serilize. Step 3: make 6-8 directional streaks using edge of loops. Step 4: make zig-zag streak in remaining space and toward the center of the plate,
Simple Stain Smear
Purpose: to stain cells and observe morphology(study of change: Chains, Clusters, Pairs) (shape) & arrangement. Stain= solvent (e.g. H20, Alcohol) + chromagen (color molecule: benzene derivative) *procedure give certain results. Solvent-compound liquid that does the mixing. Chromophore: colored portion of chromogen. Simple Stain (basic/alkaline) stain (dye): (positive charge) eg: e.coli Why use alkaline dye? - Cells have a negative charge. 4 Basic Alkaline/basic stains (+) 1. Metholyene blue 2. Crystal Violet 3. Safrain (Red) 4. Malachite Green
Why is Sabouraud Dextroses Agar used in this experiement?
Sabouraud Dextrose Agar, which has a ph of 5.6 is used to isolate fungi that outgrows most bacteria + this PH. -selective medium- are designed to suppress the growth of unwanted bacteria and encourage the desired microbes.
Endospore
Some bacterial spcies are able to differentiate into dormant cells called endospores when the environmental conditions, such as nurtrient depletion or high temperatures are unsuitable for growth. Endospores are highly resistant to heat and chemicals, which allow them to survive in this state for long periods of time. The total absence of ATP within endospores is an indication of how dormant they are. In addition to nutrient depletion, sporulation has also been shown to dependent on propulation density. With increasing density, a secreted peptide (called "competence and sporulation factor," or CSF) reaches a critical concentration and results in derepression of sporulation genes, which is a fancy way of saying, " take the brakes off" of them. Endospores resistance is due to a combination of factors, including a tough covering made of protein keratin, its dehydrated state, DNA protective proteins and other adaptations. When conditions are suitable again, endospores germinate into metabolically active vegetative cells. The Keratin in the spore coat also resists staining, so extreme measures must be taken to stain an endospore. In the Schaeffer_fulton method, a primary stain of malchite green is forced into the spore by steaming the bacterial emulsion. alternatively, malachite green can be left on the slide for 15 mins or more to stain the spores. Malachite green is a water soluble and has a low affinity for cellular material, so vegetative cells and spore mother cells (which are responsible for producting the endospore) can decolorize with water and counterstained with safranin. Endospores my be located in the middle of the cell (central), at the end of the cell (terminal), or between the end and middle of the cell (subterminal). Endospores location in some species is variable and my be combination of terminal and subterminal, for instance. Endospores also my be differentiated based on shape- either spherical, or elliptical oval-and size relative to or not. The endospore stain is a differential stain used to detect the presence, shape, and locatioin of endospres in bacterial cells. Only a few genera produce spores. Most common are the genera Bacillus (over 100 species are also endospore producers. These include Beviballus, Geobacillus, Paenibacillus, and Virgibacillus, among others. Most Bacillus species are soil, freshwater, or marine saprophytes, but two are pathogens, B anthracis is the causal agent of anthrax and B. Cereus causes two kinds of Food poisoning:emetic and diarrheal. most members of Clostridum are soil or aquatic saprophates or inhabitants of human intestines but four pathogens are fairly well known: C. Tetani (tentanus, C. botulinum (botulism, C. perfringens (gas gangrene), and C difficile (pseudomembranous colitis).
Escherichia
The bacterial species Escherichia Coli is one of the most common inhabitants of the human intestinal tract and is probably the most familiar organism in microbiology. Recall from the previous chapters that a great deal is known about the bio chemistry and genetics. of E.coli and it continues to be an important tool for basic biological research-many researchers consider it almost a laboratory pet. Its presence in water or food is an indication of fecal contamination. E.coli is not usually pathogentic. However, it can be a cause of urinary tract infections, and certain strains produced enterotoxins that cause travelers diarrhea and occassionally cause very serious foodborne diseases.
The ability to resist or not resist decolorization in the gram stain is based on:
The cell walls having a thick or thin pepridoglycan layer.
Why do bacterial cells remain unstained in a negative stain.
The negative charge on the cells surface repels the negatively charged chromogen, so the cell remains unstained against a colored background.
Gram Stains
The gram stain is a differential stain in which a decolorization step occurs between the application of two basic stains. The gram stain has many variations, but they all work in basically the same way. The primary stain is Crystal viole. Iodine is added as a mordant to enhace crystal violet staining by forming a crystal violet iodine complex. Decolorization follows and is the most cirtical step in the procedure, gram-negative cells are decolorized by the solution (generally and alcohol/acetone mixture of varying proportions) whereas gram-poritive cells are not. Gram negative cell can thus be colorized by a red counterstain safranin, but- gram positive cells are already violet and cannot. Upon sucessful completion of a gram stain, gram-positive cells appear purple and gram negative cells appear reddish-pink. Electron microscopy and other evidence indicate that the ability to resist decolorization or not is based on the different wall constructions of Gram-positive and Gram-negative cells. Gram negative cell walls have a higher lipid content (because of there outer membrane) and a thinner peptidoglycan layer than gram-positive cell walls. The Alcohol/acetone in the decolorizer extracts the lipid, making the gram-negative wall more porous and incapable of retaining the crystal violet-ioding complex, thereby decolorizing it. The thicker peptidoglycan and greater degree of cross-linked (because of teichoic acids) trap the crystal violet-iodine complex more effectively, making the gram-positive wall less susceptible to decolorization. Although some organisms give Gram-variable results, most variable results are consequences of poor technique. The decolorization step is the most crucial and most likely source of Gram Stain inconsistency. It is possible to overdecolorize by leaving the decolorization on too long and get reddish Gram-positive cells. It also is apossible to underdecolorize and produce purple Gram-negative cells. Neither of these situations changes the actual Gram reaction for the organism being stained. Rather, these are false results because of poor technique. A second source of poor Gram stains is inconsistency in preparation of emulsion. Remember a good emulsion is about a dime size and dries to a faint haze on the slide. A third source of inconsistent Gram Stain may be the organism themselves. some gram positives, especially Bacillus and straphylococcus species, lose their ability to retain the crystal violet iodine complex in as little as 24 hours of incubation. Always plan on doing your gram stains on cultures no older than 24 hours for best results. Until correct results are obtained consistently, it is recommended that control smears of Gram-positive and Gram-negative organisms be stained along with the organism be stained along with the organism in question.
Why do we use a needle when making a smear from a slant culture instead of using a loop?
The inoculation needle is used so we don't collect to much of the samples.
Negative Stains
The negative staining technigue uses a dye solution in which the chromogen is acidic and carries a negative charge. (An acidic chromogen gives a hydrogen iion, which leaves it with a negative charge.) The negative charge on the bacterial surface repels the negative chromogen, so the cell remins unstained against a colored back ground. The negative staining technique is used to determine morphology and cellular arrangement in bacteria that are to delicate to withstand heat-fixing. A primary emaple is the spirochete Treponema, which is distorted by heat fixing of other staining techniques. Also, where determining the accurate size is crucial, a negative stain can be used because it produces minimal cell shrinkage.
Negative Stains
The negative staining technique uses a dye solution in which the chromogen is acidic and carries a netagive charge. The negative charge on the bacterial surface repels the negatively charged chromogen so the cell reamins.
Ompg
The ompg test is used to differentiate late lactose fermenters from lactose non-fermenters in the family Enterobacteriacea. P. Vulgares.
Acid Fast
The presence of mycolic acids in the cell walls of acidfast organisms is the cytological basis for the acid-fast differential stain. Mycolic Acid is waxy substance that gives acid-fast cells a high affinity for the primary stain and resistance to decolorization by acid alcohol solution. A variety of Acid-fast staining procedures are employed, two of which are the Ziehl-Neelsen (ZN) method and the Kinyoun (K) method. These differ primarily in the ZN method uses heat as part of the staining process (this is in addition to heat fixing the preparation) whereas the K method is a "cold stain" (which is still heat fixed). In both protocols the bacteria smear may be prepared in a drop of serum to help the "slippery" acid fast cells (they are waxy, after all) adhere to the slide. The two methods provide comparable results and the and the choice method comes down to lab conventions and personal preferences. The waxy wall of acid-fast cell repels typical aqueous stains. (As a result, most acid fast positive orgranisms are only weakly gram positive). In the ZN method, the phenolic compound carbolfuschsin isused as the primary stain because it is lipid soluble and penetrates the waxy cell wall. Staining by carbolfuschsin is futher enhaced by steam heatingthe prepartion th melt the wa and allow the stain to move into the wall. Acid Alcohol is used to decolorize nonacid fast cells, acid fast cells resist this decolorization. A counterstain, usch as methylene blue, then is applied. Acid fats cells are reddish purple; nonacidfast cells are blue. The kinyoun method uses a slighly more liped-souluable and concentration carbolfusion as the primary stainf. These properties allow the stain to be penetrated the acidfast walls without the use of heat but makie this method slighly less sensitive than the ZN method. Decolorization with acid alcohol is followed by a contrasting counterstain, such as brilliant green. The Acid Fast is a differential stain used to dected cells capable of retaining a primary stain when treated with an acid alcohol. It is an important differential stain used to identify bacteria in the genus Mycobacterium, some of which are pathogens. (eg, M. leprae, M. Tuberculosis.
Spirocheates
The spriochetes have a coiled morphology, resembling a metal spring; some are more tightly coiled than others. The most distinctive characteristic of this order, however is their method of molity, which makes use of two or more axial filaments( or endoflagella) enclosed in a space between an outer sheath and the body of the cell. One end of each axial filament is attached near a pole of the cell. by rotating its axial filament, the cell rotates in the opposite direction, like a corkscrew, which is very efficient in moving the orgranism through liquids. For Bacteria, this is more difficult than it might seem, At the scale of bacterium, water is as viscous as molasses is to humans. However, a bacterium can typically move about 100 times it body lenght in seconds, whearas a large, fast fish, such as tuna, can move only about 10 times its body lenght in this time. Treponmea: The spirochetes include a number of important pathogentic bacteria. The best known is the genus Treponema, which includes treponema pallidum the cause of syphilis. Borrelia: Member of the genus Borrelia cause relapsing fever and lyme disease serious diseases that usually transmitted by ticks or lice.
Standard Plate Count
The viable count is one method of determining the density of a microbial population. It provides an estimate of actual living cells in a sample. Escherichia Coli.
What do gram positive cells look like after the decolorization step in the gram stain? Why?
They appear to be purple because of the Crystal Violet
Why are you asked to gram stain a mixture of gram positive and gram negative bacteria?
To obtain correct results.
Streak Plate
To obtain pure sample--->---->pure Staphyococcus epidermis, Micrococcus Luteus.
What do gram negative and gram positive cells look like before crystal violet is added in the gram stain?
Transparent.
What would happen to gram negative cells if you did not add enough alcohol in the gram stain?
Undercolorization and produces purple gram negative cells.
What are two general catergories of fungi and what characteristic are associated with them?
Yeast A. Budding yeast. B. Psuedohypha- buds that fail to detach themselves. C. Fission yeast- divide evenly to produce new cells.
Reading the PR Broth
Yellow-> acidic->PH 7 Red->neutral->Ph 7 Pink->basic-> PH>7
Simple Stain
aqueous solution of a single dye and the dye helps to highlight the organism cellular shape and structure.
Simple Stain
aqueous solution of a single dye and the dye helps to highlight the organisms cellular shapes and structure.
Saccharomyces cervesiase
bakers yeast, brewes yeast.
Rhizopus stoloinifer
causes black bread mold. A. Sporangium (sporangiospores) B. Aerial Hyphae (aka: sporangiospore) for travel. C. Aspectic hyphae (no wall adjacent to cells). D. Roots-Rhiziods- Anchor organism. coenocitic-multi-nucleated.
Eukaryotic
has a nucleus
Negative Stain
is used to determine, morphology, and cellular arrangement in bacteria that are to delicate to with stand heat-fixing. (Bacillus cereus, Micrococcus Luteus, Rhodospirillum rubrum.
Why is it usually necessary to stain bacterial cells in order to see them?
makes them more visible under microscope.
Kingdom Fungi
mycology: study of fungi eukaryotic (has nuclues) non-photosynthetic cell walls made of chitin not celluose. absorbative heterotrophs (excreates digestive enzymes, outide eats it) two division: yeats, molds. Candid Albican - found in respritory, gastro intestinal track, female urogentital track. -predoseposing factors: P.H. hormone levels, temperature salinity. - leads to thrush- abnormal growth in cavity, yeast, vulovaginitis. endocraditis, systemic (blood) infections. Saccharomyce cervesial- bakers yeast, brewers yeast AKA bakers yeast. Mold-Multicellular -procreates via binary fission -contains hyphae or individual funal filaments. * looks cottonish. - produces " spores" (asexual, sexual) Rhizopus Stoloinifer -causes bread mold. 1. Sporangium (spongiospores)- asexual. 2. Aerial Hyphae (aka: sporgangious for travel. 3. Aspectic Hype (no walls adjacent cells) 4. Roots-Rhizoids- anchor orgranism - coenocitic- multi-nucleated. Penicillium Notatium -Produces Penicillin use in cheese production. -green powdery white apron. Aperillas Niger -green yellow, brown gradular colonies with white edge. - asexual -condiospores -philides -condiospores.
The chromogen is a negative stain carries a________ charge. Why?
negative charge. An Acidic chromogen gives up a hydrogen ion, which leaves it with a negative charge.
What are stains? What do they Consist of?
staining simply means coloring the microorgranism with a dye that emphasizes certain structures. (must be heat fixed) 1. smear- thin film of material, Bunsen burners a couple times. 2. Fixed- to attach the microbe to the slide and kill the microbe.
Bacillus Anthracis
the bacillus anthracis, the bacterium that causes anthrax in animals. The endospore-forming bacillus is a large, areobic, gram postive microorganism that is apparently able to grow slowly in soil types having specfic moisture conditions. The endospores are ingested along with grasses, causing a fulmination, fatal sepsis. The incident of human anthrax is now rare in the united states, but occurances in grazing animals, hides, wool and other animals products from certain foreign countries. Infections by endospores are initiated by endospores. Once introduced into the body they are taken up by macrophages where they germninate into vegetative cells. these are not killed, but muiltiply, eventually killing macrophage. The released bacteria then enter the bloodstream, replicate rapidly and secrete toxins. The primary virulence factor of B.athracis are two extoxins. Both toxins share a third toxic component, a cell receptor binding protien call the protective antigen, that binds the toxins to target cells and permits their entry. One toxin, the endema toxin to target cells and permits their entry. One toxin, the edema toxins causes local edema (swelling) and interferes with phagocytosis by macrophages. The other toxin, lethal toxin, specifically targets and kills macrophages, which disables an essentail defense of the host. Futhermore, the capsule of B anthracis is very unusual. It is not a polysaccharide but rather is composed of amino acid residues, which for some do not stimulate a protective response by the immune system. Therefore, once the anthrax bacteria enters the bloodstream, they proliferate without any effective inhibition until there are tens of millions per milliliter. These immense populations of toxin-secreating bacteria ultimately kill the host.
Simple Stain
to stain cells and study the shape and morphology. Cheek cells, Bacillus Cerillus.
Aspectic Technique
to handle bacteria/cultures and prevent contamination for occuring. Citro Bacteria Frundill (CF), Staphyloccus (SE).
Gram Stain
used to distinguish between gram positive and gram negative cells and study the shape, morphology, size arrangement. (Staphylococcus epidermisdis, Escherichia coli, Neisseria sicca, Corynebacterium xerosis.
