Lab Practical 1 Flashcards
Rods. Can have rounded, flat, or tapered ends. Can be motile or non-motile.
Bacilli
Bacteria may occur singly, in chains, in a tetrad, or irregular masses. Most are nonmotile because they lack flagella
Cocci (spherical)
Bacteria are motile, using axial filaments, which are a type of flagella that originate form both ends of the cell and wrap around the cell body. Rotation of these causes the cell to move in a cork screwlike motion.
Spiral or curved
Agar plates
Incubated upside down to prevent moisture from collecting on the cultures and ruining them, spreading the inoculated organisms around. Agar-agar, the solidifying agent, becomes liquid when boiled and solid at about 42C.
Transfer from broth to broth
TSB of E. coli. Use a loop, swirl in the culture, swirl in the in sterile broth, flame the loop. 37C.
Slant to slant
TSA slant to sterile agar slant. E. coli. Use loop to draw culture, streak surface of sterile slant in a serpentine manner, flame loop
Plate to slant
TSA plate, slant, bacterial colonies. Use a needle. Raise the lid of the plate only a little to prevent contamination, touching the center of the colony with the needle. Streak the slant with the needle, not gouging it. Flame
Endospores require what to die?
Sterilization in the autoclave at 121C for 15-20 minutes, at 15 pounds per square inch.
What is used to clean living tissue? Surfaces?
Antiseptic is for living tissues. Disinfectant is for surfaces (leave covered for 20 minutes on a spill)
Hospital acquired infections are?
Nosocomial infections
Part of the microscope that varies intensity of light. Part of the microscope that reduces the intensity of light below the lower limit allowed by voltage control.
Light intensity control
Neutral density filter
Explain the objective lenses
10x is yellow, and 20x is green (coarse adjustment). 40x is blue, and 100x is white (fine adjustment). White is the oil immersion lens. Low power, high dry, oil immersion.
The resolving power of the microscope is a function of
The numerical aperture of the lenses and the wavelength of the light.
The ability of a lens to completely separate two objects in a microscopic field.
Resolving power, on most microscopes 1000x. It’s a function of the wavelength of light and the numerical aperture. Limit of resolution is typically 0.2um. Objects closer than that would not be seen as tow distinct objects.
A mathematical expression that describes how the condenser lens concentrates and focuses the light rays from the light source.
Numerical aperture. Its value is maximized when the light rays are focused into a cone of light that then passes through the specimen into the objective lens. The greater the loss of refracted light, the lower the numerical aperture. 0.2um
Oil immersion
This oil has the same refractive index as glass. When used it forms a continuous system that limits the loss of light due to refraction. Malaria caused by the Plasmodium species.
What microscopy is used for transparent delicate living organisms?
Which pathogenic microbe?
Darkfield. The darkfield stop is placed below the condenser, so that only oblique rays strike the objects being viewed.
Used to identify a spirochaetes in exudates from syphilitic lesions.
Goals of making a smear
Cause the cells to adhere to the slide so that they aren’t washed off during the staining and washing. Also makes sure that shrinkage of cells doesn’t occur during staining, so that distortion doesn’t result. Prepare thin smears, because thick smears obscure details and entrap the stain
Heat fixation
Allow the slide to air dry first. Then pass through the flame a few times, not leaving it too long or else the slide will shatter. It fixes organisms to the slide and kills anything that’s still living. Used in simple staining, gram staining, and spore staining.
Simple staining
The use of a single stain to color a bacterial cell. Uses S. aureus and E. coli. Bacterial cells are negative. Basic dyes are cationic (+) because of the chromophores, color-bearing ions. Methylene blue (!), basic fuchsin, crystal violet. Can determine morphology, size, and arrangement.
Negative stains
Uses B. megaterium. They are acidic (-) anionic with a chromophore that does not penetrate the cell but is rather repelled by the negative bacterial cell. Negative/indirect staining because the background is stained. India ink and nigrosin. Can be used in studying morphology, capsules, no shrinkage of cells because no heat fixing, so size determinations are more accurate. Good for observing spirochaetes.
Reactions that take advantage of the that cells or structures within cells display dissimilar staining reactions that can be distinguished by the use of different dyes
Differential staining, eg gram staining
Gram stain procedure and microbes
E. coli (-) bacillus with S. aureus (+) cocci
M. catarrhalis (-) cocci with B. megaterium (+) bacillus
Primary: crystal violet, 1 minute
Mordant: gram’s iodine, 1 minute
Decolorizer: ethyl alcohol, 20-30 seconds
Counterstain: safranin, 1 minute
Spore stain overall
The bacteria of the genera Bacillus and Clostridia become endospores when they exhaust essential nutrients. They have a protein coat, or exosporium, that forms a protective barrier around the spore. Resistant to heat, radiation, acids, and many chemicals like disinfectants.
Spore stain procedure and microbes
B. megaterium (green endospore, pink vegetative cell)
Primary: Malachite green, 5 minutes over steam
Mordant: Heat from steam
Counterstain: Safranin, 30 seconds
Acid-fast stain overall
Bacteria in the genus Mycobacterium and some in Nocardia contain a waxy mycolic acid in their cell walls. Heat allows the stain to penetrate the cell walls by softening the mycolic acid. These bacteria are acid-fast. Used to diagnose M. tuberculosis and M. leprae.
Acid-fast procedure and bacteria
M. smegmatis is acid-fast, pink. S. aureus is not, blue. Primary: carbolfuchsin, 5 minutes Mordant: Heat over steam Decolorizer: acid-alcohol, 30 seconds Counterstain: methylene blue, 30 seconds
Contains only a single kind of organism whereas _____ contains more than one kind.
Pure culture, mixed.
Obtained using a streak plate, which dilutes the bacterial cells in a sample to an end point where a single cell divides giving rise to a pure colony.
Pure culture procedure
Used a mix of S. marcescens (red), M. luteus (yellow), and E. coli (black). Incubated at 25C for the plate and tubes.
Wet mounts and hanging mounts
Wet mounts are placed directly on the glass. It can easily dry out by evaporation, though, and is bad if observations need to be made for longer periods. So then use a hanging drop mount, which has jelly that makes a glass chamber to prevent drying.
S. aureus is non-motile, P. mirabilis is motile.
False motility movement due to the molecular bombardment of cells, causing them to shake but not move in a vectoral way. Currents are created under the cover glass when the wet mount dries out or the objective touches it.
Brownian motion
Determining motility using a semisoft agar medium
Has an agar concentration of 0.4% which allows them to move. TTC is added to facilitate the direction of motility. This is where they’re stabbed into the medium using a needle. Used for pathogenic bacteria because it’s safer than wet mounts.
Bacteria that must grow in oxygen because their metabolism requires it.
Obligate aerobes, eg P. aeruginosa
Aerobic bacteria that prefer to grow in oxygen concentrations of 2-10% rather than the 20% found in the atmosphere.
Microaerophiles
Bacteria that grow very well aerobically but also have the capacity to grow anaerobically if oxygen isn’t present
Facultative anaerobes. They can carry out respiration or switch to fermentation. E. coli
Tolerate oxygen and even grow in its presence but do not require oxygen for energy production. Use strictly fermentation.
Cannot tolerate oxygen and must be cultured under conditions in which oxygen is completely eliminated
Aerotolerant anaerobes, obligate fermenters
Obligate anerobes
C. sporogenes
Medium that contains a chemical that reacts with oxygen to create anaerobic conditions
Fluid thioglycollate medium (FTM). Contains the dye resazurin, which indicates oxygen by turning pink. The medium will be pink at the top and colorless in the middle and bottom.
Obligate aerobes, facultative, micro, facultative, obligate anaerobes.
This can be used to create an oxygen free environment for the cultivation of anaerobes
GasPak anaerobic jar. Palladium pellets catalyze the reaction. Hydrogen generation is done by adding water to an envelope of chemicals, which removes oxygen by forming water. CO2 is also produced. Indicator strip of methylene blue becomes colorless in total absence of oxygen. 2 hours to decolorize.
Media that are designed to grow a broad spectrum of microbes. Contain a mixture of nutrients that can support the growth of a variety of life.
General purpose media, eg trypticase soy agar.
Contain complex organic substances such as blood or special growth factors and vitamins that certain species must have in order to grow. Fastidious bacteria that require these.
Enriched media, eg blood agar
Media that are formulated to prevent the growth of certain bacteria but not others
Selective media, eg salt agar, mannitol salt agar, macconkey agar
Media that are designed to display visible differences amongst microbes. Appear as variations in colony color, media color, formation of gas and precipitates.
Differential media, eg blood agar, mannitol salt agar, macconkey agar
Medium that contains a chemical that reacts with oxygen to create anaerobic conditions
Fluid thioglycollate medium (FTM). Contains the dye resazurin, which indicates oxygen by turning pink. The medium will be pink at the top and colorless in the middle and bottom.
Obligate aerobes, facultative, micro, facultative, obligate anaerobes.
This can be used to create an oxygen free environment for the cultivation of anaerobes
GasPak anaerobic jar. Palladium pellets catalyze the reaction. Hydrogen generation is done by adding water to an envelope of chemicals, which removes oxygen by forming water. CO2 is also produced. Indicator strip of methylene blue becomes colorless in total absence of oxygen. 2 hours to decolorize.
Media that are designed to grow a broad spectrum of microbes. Contain a mixture of nutrients that can support the growth of a variety of life.
General purpose media, eg trypticase soy agar.
Contain complex organic substances such as blood or special growth factors and vitamins that certain species must have in order to grow. Fastidious bacteria that require these.
Enriched media, eg blood agar
Media that are formulated to prevent the growth of certain bacteria but not others
Selective media, eg salt agar, mannitol salt agar, macconkey agar
Media that are designed to display visible differences amongst microbes. Appear as variations in colony color, media color, formation of gas and precipitates.
Differential media, eg blood agar, mannitol salt agar, macconkey agar
Enriched and differential medium made by adding sterile sheep blood to a sterile agar base, used to grow fastidious bacteria.
Blood agar. Beta hemolysis is complete, giving a clear zone with a clean edge around the colony. S. aureus
Alpha is incomplete, with a cloudy zone of greening around the colony because methohemoglobin. Browning. E. coli, S. enterica.
Gamma is no hemolysis or change in blood around the colony. S. epidermidis
A selective medium containing a high concentration of salt which inhibits most microbes. TSA with 10% sodium chloride.
Salt agar. Only facultative and obligate halophiles will grow in it.
S. aureus and S. epidermidis
A selective and differential medium that contains a carbohydrate that gives it its name, 7.5% NaCl, and pH indicator phenol red.
Mannitol Salt agar. Organisms that can ferment the mannitol will produce acids that will cause a color change in the media due to the phenol. S. aureus is a fermenter, yellow growth. S. epidermidis is not a fermenter, pink or white growth.
Selective and differential medium that contains carb lactose, bile salts, and the pH indicator neutral red.
MacConkey agar. Organisms that can ferment lactose will produce acids which will change the color of the colony. Selective for gram-negative organisms due to the bile salts.
E. coli fermenter, purple. S. enterica grew but was colorless.
Refers to the uptake of naked DNA molecules by bacterial cells, resulting in the expression of new phenotypic traits.
Bacterial transformation. Bacteria that uptake the DNA are called competent. The plasmid of human insulin genes were introduced into E. coli, so that strains of the bacterium now produce humulin.
Bacterial transformation procedure
Cells are transformed with the plasmid pGREEN, which confers ampicillin resistance, and has a GFP gene which codes for a green fluorescent protein. Only expressed in the presence of ampicillin. Only one that grew was +pGREEN LBA/ampicillin.
