Diagnostic microbiology Flashcards
Why is isolation of pure culture important in diagnostic microbiology?
Microorganisms in nature exist as mixed cultures, making them difficult to study. Isolation allows for the characterization of individual species from a clinical specimen.
What are the three methods of inoculation for isolating microorganisms?
- Streak plate method
- Spread plate method
- Pour plate method
What factors determine the type of culture media used to isolate microorganisms?
- Source of the sample
- Suspected species in the sample
- Nutritional requirements of the suspected organisms
At what temperature are inoculated media typically incubated to allow microorganism growth?
37ºC.
What are the methods for short-term and long-term preservation of pure cultures?
- Short-term: Refrigeration (4-10ºC)
- Long-term:
1. Frozen in liquid nitrogen (-196ºC)
2. Frozen in special freezers (-70ºC to -120ºC)
3. Lyophilization (freeze-drying)
What happens when microorganisms grow and divide on inoculated media?
Colonies form, and each colony originates from a single cell, making it identical in all cells.
How can different microbial species be distinguished in a mixed culture?
Different species have unique colonial morphologies that can be used for differentiation.
What two methods are used to identify microorganisms after obtaining a pure culture?
- Colonial morphology
- Cellular morphology (requires microscopy)
Why do microbial cells and viruses require a microscope for examination?
Their size is in the range of nanometers to micrometers, which requires magnification to be visible.
What is the difference between a light microscope and an electron microscope?
- Light microscope: Uses lenses and light to view specimens.
- Electron microscope: Uses a beam of electrons and magnetic fields for magnification.
What is the basic protocol for staining microorganisms?
- Prepare a thin smear of the specimen and air dry.
- Fix the smear by heating it.
- Stain with one or more dyes before microscopy.
What is the difference between simple staining and differential staining?
- Simple staining: Uses one dye to color all microorganisms, showing size, shape, and arrangement.
- Differential staining: Uses multiple dyes to differentiate between different types of microorganisms or cellular parts.
Give an example of a differential staining technique and its purpose.
- Acid-fast stain: Differentiates acid-fast bacteria (e.g., Mycobacterium) from non-acid-fast bacteria.
- Gram stain: Classifies bacteria into Gram-positive or Gram-negative groups.
What is the function of the bacterial cell wall?
The cell wall is a rigid structure that gives the bacterial cell its characteristic shape and is essential for growth and division.
How does the Gram-negative cell wall differ from the Gram-positive cell wall?
The Gram-negative cell wall is thinner and more complex than the Gram-positive cell wall.
What is peptidoglycan and its role in eubacteria?
Peptidoglycan is a rigid material in eubacteria that determines the shape of the cell and is a key component of the cell wall.
Do archaeobacteria have peptidoglycan in their cell walls?
No, archaeobacteria do not have peptidoglycan in their cell walls.
What is the main feature of a Gram-positive cell wall?
It has a thick layer of peptidoglycan, and teichoic acids are often attached to help transport positive ions and store phosphorus.
What is the role of the outer membrane in Gram-negative cells?
The outer membrane acts as a selective barrier based on the size and charge of molecules.
What structures are found in the Gram-negative cell wall?
It has an outer membrane, a thin layer of peptidoglycan in the periplasmic space, lipoproteins anchoring the outer membrane, and lipopolysaccharides (LPS).
How does the structure of the cell wall affect Gram staining?
The difference in cell wall structure results in different staining responses; ethanol increases permeability in Gram-negative cells but shrinks pores in Gram-positive cells.
How is endospore staining performed?
Malachite green is applied with heat to penetrate spores, followed by counter-staining with safranin.
How is capsule staining done?
Copper sulfate is used to treat the specimen, followed by staining, allowing the capsule to appear as a clear zone around the cells.
What is involved in flagella staining?
A mordant is used to thicken flagella, which is then stained for visualization.
What is fluorescence microscopy used for?
It is used to reveal specific objects in a specimen by using a dye that fluoresces at a specific wavelength.
How does immunofluorescence work in fluorescence microscopy?
A fluorescent dye is attached to a specific antibody, which binds to microorganisms in a specimen, causing them to fluoresce under appropriate conditions.
What is required for transmission electron microscopy (TEM)?
Staining with heavy metals or slicing the specimen into thin sections.
What is the main advantage of electron microscopy over light microscopy?
Electron microscopy has a shorter wavelength, providing greater resolving power (0.003 um) and can achieve magnification up to 1 million times.
How does scanning electron microscopy (SEM) work?
It generates a 3D image of the cell surface of microorganisms coated with a fine metal film.
Why is studying the detailed morphology of microorganisms important?
It helps with classification and understanding how cells respond to their environment, such as how extracellular structures like capsules can affect pathogenicity.
Why do bacteria have a high rate of metabolism, growth, and replication?
Bacteria are small and have a high ratio of surface area to volume, which allows for more efficient nutrient entry and supports a high metabolic rate.
What is the division time of a non-pathogenic strain of Escherichia coli?
approximately 20 minutes
What are pleiomorphic organisms?
Pleiomorphic organisms are bacteria that can change their cell shape as they grow.
What are the three basic shapes of bacteria?
- Coccus: Spherical cells, sometimes ovoid.
- Bacillus: Cylindrical or rod-shaped cells.
- Spirillum: Spiral or helical-shaped cells (corkscrew-like).
What is the function of enrichment media?
Enrichment media is used to increase the number of a specific microorganism in a sample by promoting the growth of the target species.
How do bacterial cells often arrange themselves?
- Diplococcus: Pairs (e.g., Neisseria gonorrheae)
- Streptococcal: Chains (e.g., Streptococcus spp.)
- Tetrads: Groups of four (e.g., Pediococcus)
- Sarcinae: Cubical packets of eight (e.g., Sarcina spp.)
- Staphylococcal: Grape-like clusters (e.g., Staphylococcus spp.)
What is the difference between chemically defined and undefined media?
Chemically defined media: Exact composition known.
Undefined media: Contains natural products like blood or beef extract.
What does “gross morphology” refer to in bacterial cells?
Gross morphology refers to the size, shape, and arrangement of bacterial cells.
What percentage of agar is typically used to solidify media?
1.5%
What does “fastidious” mean in relation to microorganisms?
Fastidious organisms have complex, demanding nutritional requirements and need undefined media for cultivation.
How is media prepared for growing anaerobes?
Anaerobes, which tolerate little or no oxygen, are sometimes grown deep in agar or in tall test tubes to limit exposure to oxygen.
Give an example of selective media.
Brilliant green agar inhibits Gram-positive bacteria and is used to isolate Salmonella species from food samples.
What is the purpose of selective media?
Selective media is designed to enhance the growth of one type of microorganism while suppressing the growth of others.
What is the purpose of differential media?
Differential media is used to differentiate organisms based on their nutritional requirements and characteristic appearance (e.g., hemolysis patterns).
What is an example of selective/differential media used in public health microbiology?
MacConkey medium inhibits Gram-positive bacteria while allowing Gram-negative bacteria to grow, and it differentiates them based on lactose fermentation.
What is tissue culture media used for?
Tissue culture media is used to cultivate plant or animal cells and is essential for growing viruses in vitro.
What is the optimal growth temperature for microorganisms?
The optimum growth temperature is the temperature at which a species grows most rapidly.
Can bacteria grow over a wide temperature range?
Yes, bacteria can grow over a wide range of temperatures. For example, Bacillus subtilis can grow from 8ºC to 53ºC.
What are the three cardinal temperatures for microorganisms?
- Minimum temperature: Lowest temperature at which growth occurs.
- Optimum temperature: Temperature at which growth is most rapid.
- Maximum temperature: Highest temperature at which growth occurs.
How do cardinal temperatures change based on the growth medium?
Cardinal temperatures can change depending on the nutritional content of the growth medium.
How does temperature affect enzyme activity in microorganisms?
Enzyme activity increases with temperature until the upper limit is reached, at which point enzymes degrade.
What are the three major groups of microorganisms based on optimum growth temperature?
- Psychrophiles: Grow best at 15-20ºC.
- Mesophiles: Grow best at 25-40ºC.
- Thermophiles: Grow best at 50-60ºC.
Where are psychrophiles commonly found, and what is their optimal temperature range?
Psychrophiles grow best at 15-20ºC and are commonly found in cold environments like oceans and the Polar Regions.
What is the optimal temperature range for mesophiles, and where are they commonly found?
Mesophiles grow best between 25-40ºC and include most microorganisms, such as those that cause diseases in humans.
What is the optimal temperature range for thermophiles, and where are they commonly found?
Thermophiles grow best between 50-60ºC and are found in hot environments like volcanic areas and compost heaps.
What is the oxygen requirement for aerobic microorganisms?
Aerobic microorganisms grow in an atmosphere with 21% oxygen and require more energy than anaerobes.
How do anaerobic microorganisms differ from aerobic ones in terms of oxygen?
Anaerobic microorganisms cannot grow in the presence of oxygen and do not use oxygen for energy-yielding reactions. They may be poisoned by oxygen.
What is the oxygen requirement for microaerophilic microorganisms?
an oxygen concentration of 1-15%.
What is the characteristic of facultative microorganisms?
Facultative microorganisms can grow in both aerobic and anaerobic conditions, utilizing oxygen when available.
What is the optimum pH range for most bacteria?
6 and 8.
What is the pH range for molds and yeasts compared to bacteria?
broader pH range, with an optimum pH around 5-6.
How does osmotic pressure affect microbial cells?
In a hypertonic solution, cells lose water and shrink; in a hypotonic solution, cells take in water and may burst; in an isotonic solution, water balance is maintained, allowing normal growth.
How do microorganisms maintain their intracellular pH?
constant internal pH (~7.5) by expelling or taking up hydrogen ions.
How can waste products affect the pH of the growth medium?
Microorganisms release acidic or alkaline waste products, and without buffering, this can shift the pH, inhibiting growth.
How do water and light affect microorganisms?
Water is essential for most microorganisms, and light is important for specific types of organisms that require it for growth or metabolic processes.
