Organisms used in Negative Staining lab
Micrococcus luteus, Bacillus cereus, Aquaspirillum itersonii
Negative staining procedure
place a drop of nigrosin at one end, place a loopful of the inoculum into the drop of stain and mix with the loop, place a slide against the drop at a 45o and allow the drop to spread along the edge of the slide, push the slide away from the drop forming a thin smear, air dry and examine
using an acidic stain that is negatively charged, so will not penetrate the cells, leaving the unstained cells easy to see against the colored background
Practical applications (2) of negative staining
-Natural size and shape can be seen b/c no heat fixation and no harsh chemicals
-Possible to observe bacteria that are difficult to stain
Why can't methylene blue be used in place of nigrosin for negative staining? Explain
Methylene blue is a basic stain that is positively charged, when an acidic stain that is negatively charged such as nigrosin must be used for negative staining
Why doesn't nigrosin penetrate bacterial cells?
The nigrosin is negatively charged, just like the cell membrane of the bacteria, which means there is a repulsion between the two, it is unable to penetrate.
a solution containing nutrients- soluble low-molecular-weight substances that are frequently derived from the enzymatic degradation of complex nutrients
a liquid medium lacking a solidifying agent, can supplement with agar (solidifying agent) to get a solid or semisolid medium
-extract of seaweed, a complex carbohydrate composed mainly of galactose, acts as solidifying agent
-liquefies at 100 and solidifes at 40
requires an agar concentration of about 1.5-1.8%, gives a hardened surface on which microorganisms can be grown
Agar deep tubes
tubes allowed to harden in the upright position- used primarily for the study of the gaseous requirements of microorganisms
-an incubator used to maintain optimum temperature
-uses dry heat so must maintain a moist environment by placing a beaker of water in the incubator during growth)
Shaking waterbath (advantage and disadvantage)
-Advantage- provides a rapid and uniform transfer of heat to the culture vessel, and its agitation provides increased aeration, resulting in acceleration of growth
-Disadvantage- can be used only for cultivation of organisms in a broth medium
Explain why the following steps are essential during subculturing
-Flaming the inoculating instrument prior to and after each inoculation
to maintain sterility
Explain why the following steps are essential during subculturing: Cooling the inoculating instrument prior to obtaining the inoculum
excess heat can fixate the samples or kill the organisms
Explain why the following steps are essential during subculturing: Holding the test tube caps in the hand to form a V
There are bacteria and contamination on our hands, and we don't want to contaminate the caps or tubes
Explain why the following steps are essential during subculturing: Flaming the neck of the tubes immediately after uncapping and before recapping
If there are microbes on the neck of the tube, you don't want to accidently bring those into the nutrient base, also you don't want to spread the microbe you were incubating by spreading it through the neck
Purposes of the subculturing procedure
To separate microbes in order to do tests on them. Also, it can be used to prepare and maintain stock cultures. The procedure is done aseptically in order to reduce the risk of contamination
Explain why a straight inoculating needle is used to inoculate an agar deep tube.
the inoculum needs to be drawn out from the bottom of the tube in a straight line, along the line of insertion
There is a lack of orange-red pigmentation in some of the growth on your agar slant labeled S. marcescens. Does this necessarily indicate the presence of a contaminant? Explain.
No it doesn't mean that there is contamination. It could just meant that you failed to inoculate a bacteria. Also, it could mean that the environment was wrong for the bacteria to grow.
Upon observation of the nutrient agar slant culture, you strongly suspect that the culture is contaminated. Outline the method you would follow to ascertain whether your suspicion is justified.
You could grow the culture more to see if cultures of unknown stuff grows.
What organisms used in lab 2- Techniques for isolation of pure cultures
Serratia marcescens, micrococcus luteus, escherichia coli
essentially diluting- involves spreading a loopful of culture over the surface of an agar plate (streak and turn, streak and turn...)
requires a previously diluted mixture of microorganisms. During inoculation the cells are spread over the surface of a solid agar medium with a sterile, L-shaped bent glass rod while the Petri dish is spun on a "lazy Susan" turntable
requires a serial dilution of the mixed culture by means of a loop or pipette. The diluted inoculum is then added to a molten agar medium in a Petri dish, mixed, and allowed to solidify
Can a pure culture be prepared from a mixed-broth or a mixed-agar-slant culture? Explain.
Yes, the components from the mixed culture just need to be separate first. This can be done by doing a streak plate or diluting the mixture and using a spread-plate technique. Then when you have individual colonies you can grow them as a pure culture.
Observation of a streak-plate culture shows more growth in Quadrant 4 than in Quadrant 3. Account for this observation.
This could be due to human error, no properly sterilizing the inoculating loop or spreading some of quadrant 1 into quadrant 4. This can also be due to the fact that a wider streak is used, so if the microbe is not diluted enough, it can grow more
Why is a needle used to isolate individual colonies from a spread plate or streak plate.
With a needle less microbes are picked up. This limits to possibility of getting two different microorganisms by accident.
How can you determine if the colony that you chose to isolate is a pure culture?
The cultures that grow should all look the same, they should have the same cultural characteristics.
What microorganisms use in lab 3- Cultural Characteristics of microorganisms
Pseudomonas aeruginosa, Bacillus cereus, Micrococcus luteus, Escherichia coli, Mycobacterium smegmatis
differences in the macroscopic appearance of the growth of microorganisms on different media. Used to separate microorganisms into taxonomic groups
Nutrient agar slants- how to evaluate
-abundance of growth
Form- agar slants
-Arborescent- (treelike growth)
-Rhizoid- (rootlike growth)mooth edges)
-Beaded- (nonconfluent to semiconfluent colonies)
-Echinulate- (continuous, threadlike growth with irregular edges)
-Effuse- (thin, spreading growth)
-Filiform- (continuous, threadlike growth with smooth edges)
Form- agar plates
-Circular- (unbroken, peripheral edge)
-Irregular- (indented, peripheral edge)
-Rhizoid- (rootlike, spreading growth)
Margin- agar plates
-Entire- (sharply defined, even)
-Lobate- (marked indentations)
-Undulate- (wavy indentations)
-Serrate- (toothlike appearance)
-Filamentous- (threadlike, spreading edge)
Elevation- agar plates
-Raised- (slightly elevated)
-Convex- (dome-shaped elevation)
-Umbonate- (raised, with elevated convex central region)
Nutrient broth cultures- how to evaluate
-Uniform fine turbidity- (finely dispersed growth throughout)
-Flocculent- (flaky aggregates dispersed throughout)
-Pellicle- (thick, padlike growth on surface)
-Sediment- (concentration of growth at the bottom of broth culture may be granular, flaky, or flocculant)