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Microbiology Laboratory
Techniques and Concepts
Microbiology Lab Practical Exam Review Part
Basic Growth Media
Difference between Tryptic Soy Broth (TSB) and Tryptic Soy Agar (TSA): TSB is the liquid medium, while TSA is the all-purpose, gel-like medium. They have the same identical composition, but TSA contains agar, which is extracted from red algae.
TSA is a complex medium.
Preparation of TSA and TSB:
TSA: Weigh 30 g of TSB and 15 g of agar, add to an Erlenmeyer flask with 1000 mL of DI water and a stir bar. Mix thoroughly, autoclave, and pour into sterile petri dishes.
TSB: Weigh 30 g of TSB, add to an Erlenmeyer flask with 1000 mL of DI water, mix until dissolved, and dispense 7 mL into test tubes. Autoclave the test tubes.
Sterilization methods and their uses:
Autoclave: Heat and steam sterilization, used for liquids, media, glassware, and non-heat-sensitive materials. Filtration: Sterilization of heat-labile substances, such as antibiotic solutions and urea broth, using 0.2 μm or 20 nm filters. Ethylene oxide: Sterilization of heat-sensitive objects. UV radiation: Sterilization of surfaces.
Dry ovens: Dry heat sterilization of glassware at 160-170°C for about 2 hours.
Media storage and usage:
All-purpose media: Provide the nutritional requirements for a wide range of bacteria. Selective media: Contain compounds that promote the growth of selected types of bacteria, such as NaCl, antibiotics, dyes, and specific inhibitors. Differential media: Contain compounds that allow differentiation between closely related organisms.
Incubation: 35±2°C for 24-48 hours.
Instruments used for culture transfer:
Inoculating loop, vortex, and microincinerator.
Aseptic Technique
Definition of aseptic technique:
Limiting microbes to where they are desired, working and maintaining pure cultures.
Steps of aseptic technique:
Sterilize the inoculating loop and hold it like a pencil. Pick up the culture from the plate. Open the cap of the second tube, sterilize the lip of the tube, mix the loop with the broth. Flame the lip of the tube again, replace the cap, and flame the loop to kill any remaining bacteria before putting it back.
Streak Plate Method of Isolation
Purpose of the streak plate method: To achieve isolated colonies or colony-forming units on the plate. A colony is a visible mass of cells on a petri dish arising from a single cell.
The streaking technique creates a dilution gradient by carrying fewer cells in the subsequent areas of streaking.
Streak plate procedure:
Streak the first quadrant, flame the loop, rotate to the right and streak the second quadrant, flame the loop, rotate to the right and streak the third quadrant, flame the loop, rotate to the right and streak the fourth quadrant and the center of the plate. Tape the plate shut and incubate at 30°C or 37°C for 24 hours.
Colony Morphology
Categories of colony morphology:
Colony shape, margin (edge), elevation, color, surface, and texture.
Definitions of colony morphology categories:
Shape: Circular, irregular, punctiform (tiny). Margin: Entire (smooth), undulate (wavy), lobate (lobed), filamentous or rhizoid. Elevation: Flat, raised, convex, pulvinate (very convex), and umbonate (raised center).
Archaea (prokaryotes): Cell wall lacks peptidoglycan, 70S ribosomes, no nucleus or organelles, wider size (400 nm-200 μm), not sensitive to antibiotics.
Protists:
Eukaryotes that cannot be classified as animals, plants, or fungi.
Majority are unicellular, with a few multicellular and colonial forms.
Characteristics of Protozoans, Algae (protists), and Fungi:
Protozoans: Generally motile, non-pigmented (some are), unicellular, eukaryotic heterotrophic microorganisms, movement by pseudopodia, flagella, cilia, or gliding, some have a cytosome for food ingestion and contractile vacuoles for osmoregulation, all reproduce asexually (some sexually). Algae: Photosynthetic protists, majority belong to Green Algae (chlorophyta/chloroplastida), can be unicellular, colonial, or filamentous, green in color and contain chlorophylls a and b, examples include Volvox (colonial) and Spirogyra (filamentous).
Fungi: Non-motile eukaryotes, absorptive heterotrophs, most are saprophytes, and some are parasites of plants, animals, and humans.
Prokaryote observed in the lab:
Bacillus subtilis: Rod-shaped, small, gram-positive, flagellated for motility, no nucleus, one circular chromosome.
Slides viewed in week 2:
Bacillus subtilis: Bacteria, forms endospores, motile, gram-positive, one circular chromosome, no nucleus. Micrococcus luteus: Bacteria, non-motile, gram-positive, smallest genome, generally aerobic, no nucleus. Rhodospirillum rubrum: Bacteria, polarly flagellated for motility, gram- negative, contains chlorophyll b, circular chromosome, aerobic and anaerobic, no nucleus.
Serratia marsescens
Bacteria
Flagella for motility Gram-negative Single circular chromosome Facultative anaerobe No nucleus
Clostridium botulinum
Bacteria
Gram-positive Shorter rods Produces potent neurotoxin Indicator organism in food industry No nucleus
Saccharomyces cerevisiae
Eukarya
Yeast Unicellular Multiply by budding Ascomycete used in food industry Nucleus
Spirogyra
Eukarya
Contains chlorophyll a and b Nucleus Green algae
Amoeba proteus
Eukarya
Pseudopodia allow change in shape and motility Reproduces asexually Nucleus
Paramecium caudatum
Eukarya
Ciliated Two types of nuclei (micro and macro) Cilia to move and to create feeding currents around the cytosome Division by transverse fission Nucleus
Blot dry with a paper towel View under 100x oil immersion
Gram Stain
Gram-positive cell membrane characteristics: Have a thick peptidoglycan layer on their cell wall, 20-80 nm The crystal violet stain will not be lost during the de-colorization step due to the peptidoglycan layer, which serves as a barrier for the crystal violet The alcohol used in decolorization may also shrink the pores of the peptidoglycan layer further, preventing the release of crystal violet
Contains teichoic acid
Gram-negative cell membrane characteristics:
Have a tenfold narrower wall that is surrounded by an outer membrane consisting of lipopolysaccharides, 2-7 nm
The alcohol used in the decolorization step extracts more lipids off the outer membrane, making it more porous, thus allowing the crystal violet to be lost and the Safranin stain to be picked up easily
Gram-variable cells:
Will display both gram-positive and gram-negative cells of the same size, shape, and arrangement (e.g., Mycobacterium)
The gram-positive organism could have stained as a gram-negative due to the age of the culture (if the culture is older than 24-48 hours, the murein layer gets leaky, allowing the crystal violet to be extracted)
Gram staining procedure, purpose of each step, and cell color after each step:
Same process as in making a smear, after heat-fix: Cover the smear in crystal violet for 1 minute Rinse with DI water Cover the smear with Gram's iodine for 1 minute Rinse with DI water Decolorize with 95% ethanol for 12-15 seconds Rinse with DI water immediately Counterstain with safranin for 1 minute Rinse with DI water Observe under 100x oil immersion Crystal violet is the primary stain that sticks to the cell wall of gram- positive bacteria Gram's iodine is the mordant used to create affinity between the primary stain and the reactive substances in the cell wall 95% ethanol is used to remove crystal violet from gram-negative bacteria, as it is a good lipid solvent and dehydrating agent
Safranin is the counterstain that sticks to the cell wall of gram-negative bacteria
Common problems in gram staining:
Improper de-colorization, thick smear, slides not made from exponential phase cultures, timing, and improper microscope adjustments
Primary stain, counterstain, and mordant:
Primary stain: Crystal violet Counterstain: Safranin Mordant: Gram's iodine
Endospore Stain
Endospores: A dormant cell that some bacterial cells are able to differentiate into based on harsh environmental conditions, such as nutrient depletion or high temperatures, that are unsuitable for growth The process is called sporulation Endospores are highly resistant to heat (100 °C/212 °F), radiation, and chemicals, protected by layers of keratin, allowing them to survive in this state for long periods of time Mature endospores and free spores will appear green under the microscope after staining, whereas vegetative cells and the sporangium that surround the mature endospore will appear red Still contain ribosomes and DNA
Examples: Bacillus, Clostridium, Sporolactobacillus
Location of endospores:
They can be located in the middle of the cell (central), at the end of the cell (terminal), or between the end and the middle (subterminal)
Medium used to induce sporulation:
Nutrient Sporulation Medium (NSM) contains calcium, manganese, and magnesium
This medium can induce sporulation of bacteria due to its limited nutrients
Endospore staining procedure, purpose of each step, and cell color after each step:
Heat-fix bacteria onto a glass slide, under the fume hood Steam the bacterial slide over a beaker containing boiling water Place a paper towel strip onto the glass slide and saturate it with malachite green (this stain is potentially carcinogenic, so wear gloves) Steam for 5-10 minutes (important to penetrate the tough layers of keratin on the endospore)
Capsules are composed of mucoid polysaccharides or polypeptides that repel stains because of their neutral charge
Capsules may increase the virulence of some microbes by preventing phagocytosis
Stained and unstained components:
Often, an acidic stain like India Ink is used to stain the background Then, a basic stain like crystal violet or safranin is used to colorize the cell This means only the capsule is left unstained To make the cells adhere to the slide, a drop of serum can be added, but the cells should not be heat-fixed
Acid-Fast Stain
Acid-fast stain:
Differential stain
Characteristics of acid-fast organisms:
Mycolic acid, a waxy substance that gives cells a higher affinity for primary stains and resists de-colorization by acid alcohol, is present in the cell wall of acid-fast organisms (Mycobacterium)
Ziehl-Neelsen method components and their purpose:
Carbolfuchsin primary stain containing phenol helps dissolve the lipid of the waxy cell wall to enhance staining Addition of heat forces the dyes into the cells Acid alcohol de-colorization is resisted by acid-fast positive species Addition of the counterstain methylene blue stains acid-fast negative cells
Flagella Stain
Typical arrangements of flagella: Monotrichous: a single flagellum (polar) Peritrichous: flagella over the entire surface Amphitrichous: flagella at both ends
Lophotrichous: tufts of flagella at the end of the cell
Modification of flagella during the staining procedure:
Flagella are often hard to see through the light microscope A mordant is often used to increase the thickness of the flagella, making them easier to view
Thioglycolate Agar Deeps
Types of aerotolerance and growth location in an agar tube: Obligate Aerobes: require oxygen for respiration (grow at the top - A) Facultative Anaerobes: prefer oxygen presence, but can grow in the absence as well (will grow from the top to the bottom - C) Microaerophiles: grow at lower atmospheric oxygen tension (below the surface - D)
Obligate Anaerobes: grow where oxygen is absent, lack enzymes superoxide dismutase and catalase which break down toxic forms of oxygen H₂O₂ and O₂¯ (bottom of the media - B)
Purpose of sodium thioglycolate:
Agar Deep Stabs are prepared with TSA, sodium thioglycolate (consumes oxygen), and yeast extract to promote the growth of a broad range of organisms
If resazurin is added to the medium, a red color change indicates dissolved oxygen in the medium
Creation of the oxygen gradient:
Oxygen is removed from the medium during preparation and autoclaving, but will immediately begin diffusing back in as the medium cools This creates an oxygen gradient from aerobic at the top to anaerobic at the bottom
Anaerobic Jar
How an anaerobic jar works: The jar uses a gas-generating system that contains a packet (Gas Pak) of inorganic carbonate, activated carbon, ascorbic acid, and water When the outer wrapper is removed, the system becomes activated with the exposure to air When the jar is sealed, the atmospheric oxygen will rapidly decrease, creating an anaerobic chamber in the jar
Inorganic carbonate also produces CO₂
Interpretation of growth/no growth on a plate from the anaerobic jar:
Growth = anaerobic No growth = aerobic
Catalase Test
Purpose and enzyme tested: Catalase converts hydrogen peroxide into water and gaseous oxygen
Nitrate Reduction
Purpose and Enzyme Tested
The nitrate reduction test is used to identify bacteria that can reduce nitrate to nitrite. Many gram-negative bacteria contain the enzyme nitrate reductase, which performs this single-step reduction known as assimilative nitrate reduction. For example, Escherichia coli partially reduces nitrate to nitrite, while other bacteria like Pseudomonas and Bacillus can further reduce nitrate to N₂O and N₂ (gases).
Test Procedure
Nitrate broths are inoculated with bacteria and then incubated. If the presence of a bubble in the Durham tube is uncertain, reagents A and B are added under a fume hood for a minute. If the medium turns red, the result is positive (+). If it does not turn red, zinc is added, and if it then turns red, the result is negative (-).
Reaction and Interpretation
The nitrate broth will turn red if the nitrate reduction is positive. If the broth does not change color, the addition of zinc will turn it red, indicating a negative result. The red color is produced when the sulfanilic acid reagent reacts with the nitrous acid to form diazotized sulfanilic acid, which then reacts with the α-naphthylamine to form a red-colored compound.
Mannitol Salt Agar
Purpose and Selectivity
Mannitol Salt Agar is a differential and selective medium. It is differential because it contains the carbohydrate mannitol, which is a substrate used in fermentation, and phenol red, a pH indicator. It is selective because it contains 7.5% sodium chloride (NaCl), which is a high enough concentration to dehydrate and kill most bacteria.
Color Change and pH Levels
Phenol red is red at pH 8.2 and yellow at pH 6.8. Mannitol-fermenting bacteria will decrease the pH, making the medium a bright yellow color.
Growth Interpretation
The purpose of using this medium is to isolate bacteria that can withstand the high osmotic pressure created by the high salt concentration. Growth on the plate can be interpreted as follows:
- Little to no growth: Not Staphylococcus or Micrococcus
Good growth: Staphylococcus, Micrococcus, or Bacillus subtilis Yellow growth (halo): Staphylococcus aureus and B. subtilis Red growth: Other Staphylococcus species or Micrococcus
MacConkey Agar
Purpose and Selectivity
MacConkey Agar is a differential and selective medium. It is differential because it contains lactose, which can be fermented by some organisms. It is selective because it contains bile salts and crystal violet, which inhibit the growth of gram-positive bacteria.
Color Change and pH Levels
MacConkey Agar contains the pH indicator neutral red, which is colorless at pH above 6.8 and red at pH less than 6.8. If lactose is fermented into acid end-products, the pH will be lowered, and the medium will generate a red color.
Growth Interpretation
The purpose of using MacConkey Agar is to isolate and differentiate members of the Enterobacteriaceae family. If the medium is made without crystal violet, some Staphylococcus and Enterococcus species that ferment lactose will also grow.
The growth on the plate can be interpreted as follows:
Little or no growth: Gram-positive bacteria Good growth: Gram-negative bacteria Bright pink/red growth: Lactose-fermenting gram-negative bacteria Colorless growth: Non-lactose-fermenting gram-negative bacteria
