Lab Procedures
What is the Gram Stain Procedure?
The Gram Stain procedure involves heat fixing the bacteria to the slide,then dying it crystal violet and iodine, decolorize it with ethanol, and counterstain with safranin.
What are the different types of tubed media?
Broth, Agar slant, and Agar deep.
Name all the bacteria used during the first half of the semester and with what procedure do they coincide with.
1. Bacillus Cereus (Simple Stain, Negative Stain, Endospore stain)
2. Staphylococcus Epidermis (Simple stain & Gram stain)
3. Escherichia Coli (Gram stain)
4. Klebsiella pneumoniae (Capsule stain
5. Serratia Marcescens
6. Alcaligenes Faecalis
7. Entercococcus Faecalis
8. Enterobacter aerogenes
Explain the effects of Temperature on Microbial Growth.
With the increase of temp., it also increase enzyme activity. The following temperatures are...
1. Psychrophiles= organisms that grow under 20 degrees celsius
2. Psychrotrophs= organisms that grow 0-30 degrees celsius
3. Mesophiles= organisms that grow 15-45 degrees celsius
4. Thermophiles= organisms that grow above 40 degrees celsius
5. Extreme Thermophiles= organisms that grow 65-110 degrees celsius
Explain IMViC Test.
A group of individual test used to identify an organism in a coliform group or differentiate the enterics (Family Enterobacteriaceae)
A coliform is a gram negative, aerobic, or facultative anaerobic rad, which produces gas from lactose within 48 hrs.
What is the procedure of Capsule stain?
1. Stain slide with Congo Red I.
2. Emulsify bacteria with dye, then spread it across slide. Let completely dry (NO HEAT-FIXING)
3. Apply Congo Red II. Let stand for 1-2 min. Rinse off gently with water.
4. Blot dry and observe with microscope.
Define Selective Medium and give examples.
A medium designed to enhance Isolation and inhibit growth of some organisms but encourage others.
Some examples for selective medium is Phenylethyl Alcohol Agar (PEA), Mannitol Salt Agar (MSA), MacConkey Agar (MAC), and Eosin Methylene Blue Agar (EMB)
What are the different shapes of the bacteria?
1. Spherical (cocci)
2. Rod (bacilli)
3. Spiral (spirilla)
4. Comma (vibrios)
5. Corkscrew (Spirochaetes)
They can also exist as single cells, pairs (diplococci), chains, or clustersExplain the effect of osmotic pressure on Microbial Growth.
It draws water from the bacteria microorganisms. Since microorganisms have distinct ranges for optimal growth, once outside that range they die.
Explain Voges-Proskauer Test and the product being detected.
A test designed for organisms that are able to ferment glucose, but quickly convert their acid products to acetoin and 2, 3-butanediol (used to be detected by Barritt's reagent).
A positive result of VP is red. A negative result is when there is no color change (or development of a copper color).
What is the procedure for Endospore Stain?
1. Inoculate bacteria and heat fix to slide
2. Cover smear with Malachite green stain (the heat is used to force stain into spores). Wash off after a min.
3. Counterstain with safranin and then wash again after 1 min.
4. Blot dry and observe.
Define Differential Medium and give examples.
It's a medium that distinguishes between different microbes.
Some examples for this is Mannitol Salt Agar (MSA), MacConkey Agar (MAC), and Eosin Methylene Blue Agar (EMB).
What are the different types of colony morphology?
1. Shape= rounded, irregular, or punctiform (tiny, pinpoint)
2. Margin= entire (smooth, w/ no irregularities), undulate (wavy), lobate (lobed), filamentous/ rhizoid (branched like roots)
3. Elevations= flat, raised, convex, pulvinate (very convex), and unbonate (raised in center)
4. Texture=moist, mucoid, dry
5. Pigment= opaque, translucent, shiny, or dull
Explain the effect of pH on Microbial Growth.
pH denaturizes cellular enzymes. Once bacteria reaches out of their are of optimal growth in specific pH ranges, it destroys the cells. The three classifications for pH ranges are...
1. acidophiles= organisms adapted to ranges below 5.5
2. neutrophiles= organisms adapted to ranges between 5.5 and 8.5
3. alkaliphiles= organisms adapted to ranges above 8.5
Explain Citrate Test.
It tests for the ability of an organism to use citrate as its sole source of carbon.
What is the procedure of simple stain?
1. Place drop of water on slide, then inoculate with bacteria.
2. Heat fix the slide
3. Stain with dye crystal violet (1 min), safranin (1 min), and Methylene blue (1 min). Wash off in between staining
4. Blot dry and observe.
What are the three methods of isolation of pure culture to mixed culture?
Streak plate, spread plate, and pour plate
What is the ubiquity of microorganisms?
Ubiquity of Microorganisms are microorganisms that are present nearly everywhere.
Explain the effect of UV Radiation on Microbial Growth.
UV radiation damages DNA. It's classified in three stages...
1. UV-A= longest wavelength, 315-400 nm
2. UV-B= ranges from 280-315 nm
3. UV-C= Most detrimental to bacteria, 100-280 nm
Explain Indole Test and how the product is detected.
This test differentiates E. Coli (Indole +) and Proteus Mirabilis (Indole -) from closely related enteric bacteria and proteus species. Indole can be detected with Kovac's reagent.
What is the procedure for negative stain?
1. Place a drop of nigrosin stain on slide and inoculate with bacteria. Then spread across slide.
2. Allow slide to air dry (NO heat fixing)
3. Observe
What is the effect of culture age on presence of endospores inside the cell?
Endospore-forming a bacteria cannot uniformly grow endospores when a culture is new and growing. Instead they only grow in response to a scarcity of nutrients which can only occur in older cultures.
List 2 methods of enumeration of microorganisms and explain how to use and calculate with cfu/mL formula.
The two methods are plate counts and serial dilutions.
In order to calculate cfu/mL is to multiply the number of colonies on the plate X to how many times the sample was diluted. Then you divided that answer by the volume plated.
Explain the effect of antibiotics on Microbial Growth. What is the disc diffusion assay of antibiotic sensitivity?
Antibiotics disrupt essential processes or structures in the bacterial cell. This either kills the bacterium or slows down bacterial growth.
Disc diffusion test for Antibiotic Sensitivity testing is carried out to determine the appropriate antibiotic agent used for a particular bacterial strain isolated from clinical specimens. An effective assay will produce a large zone of inhibition (halo), while an ineffective assay won't effect bacterial growth.
Explain MPN Test and how to read the MPN table.
Most Probable Number is a method used to estimate the concentration of viable microorganisms in a sample by means of replicate liquid broth growth in ten-fold. The table enables us to calculate for a sample the microbial numbers that are statistically likely to lead to such a result.