Exercise 9 -- The Gram Stain
Compare and contrast simple and differential staining techniques. Simple Staining
Simple staining is useful in determining the basic morphology of an organism. Simple staining involves only one reagent
i.e. crystal violet, basic fuchsin, or methylene blue.
Simple dyes used to stain bacteria have a positive charge cationic (+), therefore, they are attracted to the bacteria that are usually negatively charged anionic (-) Depends on the fact that bacteria differ chemically from their surroundings and thus can be stained with their environments Simple stains are used to provide contrast between specimen and background Differential Staining
Bacteria react differently to the multiple reagents that are used i.e. Gram Stain = Crystal violet, Grams Iodine, 95% ethyl alcohol, and safranin. i.e. Acid-fast stain
Can distinguish betweeen 2 types of bacteria because bacteria differ from one another chemically and physically to react differently to a given staining procedure. State the purpose of the Gram stain.
The Gram stain separates bacteria into two major groups: Gram positives that stain purple, and Gram negatives that stain pink or red. Primary Stain = Crystal violet; Mordant = Grams Iodine; Decolorizer = 95% ethyl alcohol; Counterstain = Safranin. Perform and interpret a Gram stain.
Gram positive = stains purple
Gram negative = stains pink or red
Discuss the consequences of deviations from proper Gram staining technique. If there is deviation from gram staining, gram positive bacteria may stain and look like they are gram negative, or vice versa.
Exercise 10 -- Miscellaneous Staining
State the purpose of the acid-fast, endospore, and capsule stains. Acid-Fast Stain
Another example of a differential stain.
Used to diagnose diseases like Tuberculosis.
Stains mycobacteria which cause tuberculosis
Mycobacteria contains mycolic acid (waxy substance) in the cell wall and it makes the organism very slow-growing and thus difficult to isolate and identify. Endospore Stain
Stain bacteria with endospores such as Clostridium and Bacillus. Because of their nearly impenetrable cell walls, the Gram-stain method will not stain endospores; therefore, this specialized staining method is necessary. Schaeffer-Fulton Method uses malachite green stain with heat and safranin for a counterstain. The endospore will stain green and the surrounding vegetative cell will stain pink. Capsule Stains (Negative Stain)
To identify bacteria with capsules, since capsules do not stain with simple staining or gram-staining One method uses nigrosine (or India ink), which stains the background black. Crystal violet is used as a counterstain to stain the bacterial cell, thus making the capsule visible as a clear halo around the cell. Capsule = when slime layer is highly symmetrical and organized; play a role in the virulence (disease-causing ability) of some bacteria. Describe the purpose of endospores, and compare and contrast two common genera that produce them. Endospores allow bacteria to survive in extreme environmental conditions such as heat, cold, or drought. Survive extreme heat, lack of water, and many toxic chemicals and radiation due to them being highly durable, dehydrated cells with thick cell walls. Unique to certain genera of bacteria
Gram positive, spore forming, bacilli (rods)
Pathogenic but easier to get rid of than clostridium.
Gram positive, spore forming, bacilli (rods)
Describe the purpose of a capsule.
Gives cell its virulence factor: anything bacteria have evolved to make them more pathogenic Prevent phagocytosis which means antibodies against the bacteria are useless and ineffective.
Exercise 11 -- Culture Media Preparation
Compare and contrast the uses of general-purpose, selective and differential media, and provide examples of each. General-purpose Media
These are nonselective primary isolation media used for culturing a wide variety of microorganisms. Consists of beef extract, peptone, and agar.
Ex: Nutrient Agar and Broth, TSA plates
Allow only certain types of bacteria to grow
Usually have inhibitory substances that restrict the growth of other unwanted bacteria. Ex: Columbia (CNA) Media
Contains various substances that cause some bacteria to take on a different appearance from other species. Ex: EMB (Eosin Methylene Blue)
Calculate proportions necessary to make different amounts of culture media when given a recipe. the weight knownvolume known=the weight wantedthe volume wanted Discuss the importance of sterilization in medium preparation, state the conditions under which complete sterilization occurs, and name the equipment used to achieve it. Sterilization of culture media is done to eliminate contaminating microorganisms from the environment. Complete sterilization occurs at 250 degrees Fahrenheit (121.6 degrees Celsius) at 15 pounds per square inch (psi) of steam pressure. Autoclaves provide this type of sterilization.
Exercise 12 -- The Streak Plate and Colony Morphology
Describe the purpose and principle of the streak plate.
Used to isolate bacteria colonies and helps to obtain a pure culture for further studies. The more streaks, the more the bacteria will be diluted until, in theory, only one cell is left to grow and give rise to a colony of the same bacteria. Explain why Petri plates are incubated in an inverted position. Lessens the risk of contamination of other microbes settling on them and to prevent water condensation that might compromise a culture. Describe how colonies form on a Petri plate and explain why isolation is an important procedure in microbiology. Isolation is an important procedure in microbiology because it helps to obtain a pure culture for further studies. Without a pure culture, you would not be able to tell if the bacteria was what you really wanted to look at or not Since bacteria of the same species will produce nearly identical colonies, isolation will confirm whether or not a pure culture was obtained.
Exercise 13 -- Specimen Transport & Ubiquity of Microorganisms Describe the principle and purpose of RODAC plates, and apply the guidelines for evaluating degree of surface contamination. Used for the detection and enumeration of microorganisms present on surfaces of sanitary importance. Replicate Organism Detection & Counting
Colonies per RODAC Plate
GOOD = 0-25; FAIR = 26-50; POOR = 50 and over
Describe the importance of quality control (QC) in microbiology in general, and the Gram stain in particular Generally:
to verify a satisfactory level of freedom from contamination, To demonstrate the correct performance of the medium when used in the usual or widely accepted manner, Ensure against significant physical imperfections that may compromise the utility of the media. Gram Stain
To be sure that you stained the organism correctly
Also, to make sure that the stains are working the way they should ie: gram-positives come out purple, and gram-negatives come out pink Important when trying to identify unknowns
Exercise 14 -- Hand-Washing
State the importance of hand washing before and after microbiological procedures, including the use of disinfectant when scrubbing for a medical procedure. Washing your hands before microbiological procedures is important because you decrease the chance of contaminating any cultures you work with. After microbiological procedures, you probably have a lot of contaminants (transient bacteria) on your hands that can make you sick. Washing your hands kills some of these and decreases your chance of becoming sick afterwards. Using disinfectant when scrubbing for a medical procedure is important so that you provide a sterile environment for the procedure so that you do not cause an infection in a patient. Define the terms nosocomial, contaminant, transient and resident as they apply to microorganisms. Nosocomial Infections = infections that are spread in a hospital environment Patient did not have the infection before she came into the hospital; newly acquired from the hospital. Transient Bacteria = Contaminants that are not part of the normal human flora; may be present for a finite time. Resident Bacteria = Permanent residents of normal human flora Contaminants = Bacteria that you do not want present in a culture medium; Also, bacteria that are not part of the normal flora.
Exercise 15 -- Bacterial Plate Counts
Use the plate count technique to calculate bacterial density in a sample. Serial dilutions?
Perform serial dilutions using serological and digital pipettors. Perform dilution problems
[FINAL (CFUs/mL)] = COLONY FORMING UNITS (CFUs) / VOUME PLATED (mL) [ORIGINAL (CFUS/mL)] = [FINAL] / DILUTION FACTOR
Define the terms CFU, aliquot, dilutent, dilution factor, TNTC, and TFTC. CFU = colony forming unit
Number of colonies that you count
Must be between 30-300 colonies
Aliquot = Smaller volume withdrawn from a total sample volume. Dilutent = Fluid used to dilute a sample.
Dilution factor = Fraction by which original sample concentration is diluted. TNTC = Too Numerous To Count = Colony count is greater than 300 TFTC = Too Few To Count = Colony count is less than 30
Explain the convention of only counting plates that have between 30 and 300 colonies. Counts over 300 colonies are considered invalid because of overcrowding that may cause two or more bacteria to form a single colony; also, they are too tedious to count accurately. Counts under 30 colonies are invalid because there may have been a sampling error.
Exercise 16 -- Bacterial Growth Characteristics
A: Osmotic Pressure
Describe osmotic pressure and how it affects a cell.
Osmotic pressure is the pressure that water exerts on a cell from either leaving the cell (hypertonic conditions) or entering the cell (hypotonic conditions) The force that is exerted to maintain the concentration differences between solutions on opposite sides of the membrane. Hypertonic Conditions = water moves out of the cell causing it to crenate or shrink Hypotonic conditions = water moves into the cell causing the cell to lyse or explode Isotonic conditions = No net water movement, no osmotic pressure. Most bacteria exist at hypotonic solutions
Some can exist in hypertonic solutions = halophiles.
Describe and recognize facultative anaerobe, strict aerobe, aerotolerant anaerobe, obligate anaerobe. Facultative anaerobes
They can grow with or without oxygen, but grow better with oxygen because they can utilize it! In thioglycollate media, it will be found more towards the top (oxygen) but can still be seen throughout the tube. Strict aerobe
Cannot survive without oxygen.
Found at top of thioglycollate where oxygen is.
Cannot utilize oxygen to grow, but can tolerate it.
Found throughout the tube, but gathers more at the bottom where there is less oxygen. Obligate (Strict) Anaerobe
Cannot exist in presence of oxygen
Oxygen is toxic to them.
Found at very bottom of thioglycollate tube.
Grow best when the atmosphere has increased CO2 (carbon dioxide) and lower concentrations of oxygen are present. Found near middle of thioglycollate tube.
Know the parts of the anaerobe jar and how it works.
Used to remove oxygen from a sealed container by catalyzing the chemical combination of oxygen with hydrogen to form water. Uses either a gas pack that has sodium bicarbonate and sodium borohydride, along with the catalyst palladium or an AnaeroGen sachet. Methylene blue indicator strip is added to detect the absence of oxygen. Blue = oxygen; Red = no oxygen; Blue = oxygen, White = No oxygen C: pH
Understand why pH is important to know for food preservation methods. Certain pH levels can prevent microbial growth on food.
pH affects enzyme function/shape in microbes; if not at optimal pH, the microbe will not work like it is supposed to and will probably die. D: Temperature
Discuss why it is useful to know the temperatures at which bacteria grow. You will know what temperatures to use to avoid microbial growth. Bacteria cannot regulate their internal heat.
If different temperature is present than optimal temperature, bacteria will not grow or they will die. Know the optimum temperature of human pathogens
98.6 degrees Fahrenheit and 25-40 degrees Celsius
Define the terms thermophile, mesophile, and psychrophile.
Can grow at temperatures between 45 and 65 degrees Celsius
Grow best between 25 and 40 degrees Celsius
Same as our body temp
Grow between 0 and 5 degrees Celsius.
Describe the different types of pigment production.
Pigment spreads from microbes and changes color of the surrounding agar. Non-diffusible pigment
Pigment that only colors the colony