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Exp 9- Negative Staining Exp 1- Culture Transfer Techniques Exp. 2- Techniques for Isolation of Pure Cultures Exp. 3- Cultural Characteristics of Microorganisms Basic lab techniques

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

regent used for negative stain

Nigrosin (needs to be negatively charged)

Negative staining

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.

Pure culture

a culture containing a single unadulterated species of cells

Culture medium

a solution containing nutrients- soluble low-molecular-weight substances that are frequently derived from the enzymatic degradation of complex nutrients

Broth medium

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

What temp does agar melt and solidify

liquefies at 100C solidifies at 40C

Solid medium

requires an agar concentration of about 1.5-1.8%, gives a hardened surface on which microorganisms can be grown

Semisolid medium

forms with less than 1% agar

agar slants

allowed to cool and harden in a slanted position, used for maintaining pure cultures

Agar deep tubes

tubes allowed to harden in the upright position- used primarily for the study of the gaseous requirements of microorganisms

Agar plates

poured into petri dishes and allowed to solidify- provides large surface areas


the process of rendering a medium or material free of all forms of life


transferring microorganisms from one vessel to another

Cultivation chambers

-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

What organism used in Lab 1- culture transfer techniques

Serratia marcescens

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

cultural characteristics

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
-optical characteristics

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)

Consistency- agar slants

Dry, buttery (moist and shiny), mucoid (slimy and glistening)

Nutrient agar plates- how to evaluate

size, pigmentation, form, margin, elevation

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)

Nutrient gelatin- how to evaluate

-Crateriform- (liquefied surface area is saucer-shaped)
-Napiform- (bulbous-shaped liquefaction at surface)
-Infundibuliform- (funnel-shaped)
-Saccate- (elongated, tubular)
-Stratiform- (complete liquefaction of the upper half of the medium)

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