simple, differential staining and motility

8/12/2019 Simple, Differential Staining and Motility

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Simple Stain Results

rpose: To recognize the three basic shapes of

cterial cells.

nciple: In order to observe most bacterial cells

ng bright field microscopy the cells must be

rk enough to see, that is they must have contrast

the light. To create contrast a simple stain can

used. Simple stains use basic dyes hich are

sitively charged. These positive dyes interact

th the slightly negatively charged bacterial cell

all thus lending the color of the dye to the cell

all.

ple staining method for bacteria in milk !method of "reed#

ection of bacteria in milk sample is difficult due to the presence of fat and protein. Several

mmonly used stains are, therefore, not serving the purpose. In this method smear is treated

h %ylene to remove fat and the fi%ation is done by alcohol.

e se&uence of steps to be folloed is as under. Place a clean slide over one centimeter

are, dran on a paper and place '.'( ml of milk sample in the centre of the s&uare. Spread

sample ith needle to cover the s&uare. $ry the smear ith gentle heat and immerse the

e in %ylene or chloroform to remove fat. )i% the smear ith *+ alcohol for - minutes.

in the smear ith "reeds methylene blue !S/'#

/ minutes. 0ash the smear ith *' alcohol till smear appears faintly blue. $ry in air and

mine under oil immersion ob1ective. "acteria are seen dark blue against light blue

kground.

PRI23IP45:

Simple staining: In the first e%ercise, you ill observe bacteria sub1ected to simple staining

in order to analyze different bacteria ith respect to their size, shape and arrangement. To

this end you ill observe, 5scherichia coli, "acillus subtilis and 6ibrio sp. as ell as

Staphylococcus epidermidis and Streptococcus lactis.


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"acteria have three basic characteristic morphologies. Those are the coccus !cocci Pl#, the

bacillus !bacilli Pl# and the spiral shaped bacteria such as the spirillum, spirochetes and

6ibrio sp. The cocci are spherical shaped bacteria hile the bacilli are rod shaped bacteria.

There is a great deal of variety ith respect to the morphologies that spiral shaped bacteria

assume7 the spirillum and spirochetes appear different under observation because of the

manner in hich they move. 0hen spirillum move the long a%is of their bodies remain rig

such that they appear to bend at the ends, hen the spirochetes move, the long a%is of their

bodies are not fi%ed such that they take on a avy, 8permed hair9 appearance. 6ibrio sp. is

rod shaped bacteria that are bent near the middle such that they are often called the 8comma

shaped bacilli9. 0hen bacteria gro and divide they can stick together in a manner that aid

in their characterization ith respect to their genus and species. )or e%ample if cocci shape

bacteria divide and stick together to form pairs, they are called diplococci7 if they stick

together to form long chains they are called streptococci and if they adhere to each other to

form 8grape like9 clusters they are called staphylococci. 3ertain bacillus shaped species

such as "acillus subtilis can adhere to each other in a chain like formation knon asstreptobacilli.

ram staining: The ne%t part of the e%ercise involves using a differential staining procedur

called ram staining to distinguish beteen ram positive and ram negative bacteria. To

this end you ill stain and observe 5scherichia coli and;or Staphylococcus epidermidis

and;or "acillus subtilis in a mi%ed culture.

(. Preparing a smear from a li&uid culture:

Place a small amount of the culture on a microscope slide as shon by your instructor and

heat fi% the sample.

ou ill make a smear of the bacterial species, by aseptically transferring bacteria from a

culture tube onto the glass slide. To this end, obtain a sterile cotton sab and sab into the

test tube containing the mi%ture of bacteria. 0ring out the sab on the inner surface of thetest tube. ?ake a thin smear on the surface of your microscope slide. It is important that

you make a thin smear such that it is easier to observe individual bacteria.

@llo the smears to dry completely7 the smears should become somehat cloudy as they

dry, it is important that the smears are completely dry as the folloing heat fi%ation step i

distort the morphology of organisms in a smear that is not completely dry.


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Aeat fi% the bacteria in the smear by &uickly passing the microscope slide through a flame B

(' times.

/. 3rystal violetDprimary stains: Place the slide containing heat fi%ed smears on a test

tube rack in the sink ith the smear facing up. 3over the smear ith crystal violet and leav

the stain on for ( minute.

-. ramEs IodineDmordant:


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+. Re flame the loop making sure the entire length of the ire that ill enter the tube ha

been heated to redness

H. Remove the tube cap ith the fingers of the hand holding the loop.

. )lame the tube mouth.

B. Touch the inoculating loop to the inside of the tube to make sure it is not so hot that i

ill distort the bacterial cells7 then pick up a pinhead size sample of the bacterial groth

ithout digging into the agar.

*. Re flame the tube mouth, replace the can, and put the tube back in the holder.

('. $isperse the bacteria on the loop in the drop of ater on the slide and spread the dro

over an area the size of a dime. It should be a thin, even smear.

((. Re flame the inoculating loops to redness including the entire length that entered the

tube.

(/. @llo the smear to dry thoroughly.

(-. AeatCfi% the smear cautiously by passing the underside of the slide through the burne

flame to or three times. Test the temperature of the slide after each pass against the back

of the hand. It has been heated sufficiently hen it feels hot but can still be held against the

skin for several seconds. Fverheating ill distort the cells.

(G. Stain the smear by flooding it ith one of the staining solutions and alloing it to

remain covered ith the stain for the time designated belo.

?ethylene blue C ( minute

3rystal violet C -' seconds

3arbol fuchsin C /' seconds

$uring the staining the slide may be placed on the rack or held in the fingers.

(+. @t the end of the designated time rinse off the e%cess stain ith gently running tap

ater. Rinse thoroughly.

(H. 0ipe the back of the slide and blot the stained surface ith bibulous paper or ith a

paper toel.


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ash, and ill be of original color. The species hich retain the stain are called ram

positive, hereas those hich yield the stain to alcohol are called ramCnegative bacteria.

Then, there is applied a counter stain a dye of some contrasting colour !step G#.

The generally used counter stains are,eosin !red#, Safranin !red#, brilliant green

or "ismarck bron. 5ach of these colors

the ramCnegative species. The cells

become no clearly visible.

The reasons hy bacteria respond

differently to the ram stain are not

completely understood. It has been

postulated that since ramCnegativebacteria have relatively a high lipid

content in their cell alls, the alcohol

dissolves the lipids, that allos the

leakage of crystal violetCiodine comple%.

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The ram positive bacteria ith less lipids in their cell alls are less susceptible to the

action of alcohol. @nother theory suggests that the peptidoglycans found in high

concentration in the cell alls of ramCpositive bacteria perhaps traps the crystal violet

iodine comple% in its many cross linkage.

The ram negative bacteria ith less peptidogylycan and feer cross linkages loose the

stain readily. It is important to note that there are some characteristic differences beteen

most ramCpositive and ramCnegative bacteria. It should be evident that the property of

ram positive ness is related to very fundamental physiological properties of the cell.


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are ramCvariable !that means, they may stain either negative or positive#7 some organisms

are not susceptible to either stain used by the ram techni&ue. In a modern environmental o

molecular microbiology lab, most identification is done using genetic se&uences and other

molecular techni&ues, hich are far more specific and informationCrich than differential

staining.

Bacterial Motility

?any but not all bacteria e%hibit motility, i.e. selfCpropelled motion, under appropriate

circumstances. ?otion can be achieved by one of three mechanisms:

?ost motile bacteria move by the use of flagella !singular, flagellum#, rigid structures /' nm

in diameter and (+C/' Nm long hich protrude from the cell surface !e.g. 3hromatium#.

Spirochetes are helical bacteria hich have a specialized internal structure knon as thea%ial filament hich is responsible for rotation of the cell in a spiral fashion and conse&uen

locomotion !e.g. Rhodospirillum#.

liding bacteria all secrete copious slime, but the mechanism hich propels the cells is not

knon. eg cyanobacteria, my%obacteria.

In some bacteria, there is only a single flagellum C such cells are called monotrichous. In

these circumstances, the flagellum is usually located at one end of the cell !polar#. Some

bacteria have a single flagellum at both ends C amphitrichous. Aoever, many bacteria have

numerous flagella7 if these are located as a tuft at one end of the cell, this is described aslophotrichous !e.g. 3hromatium#, if they are distributed all over the cell, as peritrichous. Th

folloing digital video shos motile 3hromatium cells: short, ramCnegativerods, O( Nm

diameter and -CG Nm long. 0atch for the tumbles as the cells change direction.

)lagella consist of a hollo, rigid cylinder composed of a protein called flagellin, hich

forms a filament anchored to the cell by a curved structure called the hook, hich is attache

to the basal body. )lagella are, in effect, rotary motors comprising a number of

proteinaceous rings embedded in the cell all. These molecular motors are poered by the

phosporylation cascade responsible for generating energy ithin the cell. In action, the

filament rotates at speeds from /'' to more than (,''' revolutions per second, driving the

rotation of the flagellum. The organization of these structures is &uite different from that of

eukaryotic flagella. The direction of rotation determines the movement of the cell.

@nticlockise rotation of monotrichious polar flagella thrusts the cell forard ith the

flagellum trailing behind. Peritrichous cells operate in the same ay.
http://www.microbiologybytes.com/video/cilia.htmlhttp://www.microbiologybytes.com/video/Gram.htmlhttp://www.microbiologybytes.com/video/cilia.htmlhttp://www.microbiologybytes.com/video/Gram.html


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Periodically the direction of rotation is briefly reversed, causing hat is knon as a

tumble, and results in reorientation of the cell. 0hen anticlockise rotation is resumed,

the cell moves off in a ne direction. This ability is important, since it allos bacteria to

change direction. "acteria can sense nutrient molecules such as sugars or amino acids and

move toards them C a process is knon as chemo ta%is. @dditionally, they can also move

aay from harmful substances such as aste products and in response to temperature, light

gravity, etc. This apparently intelligent behavior is achieved by changes in the fre&uency of

tumbles. 0hen moving toards a favorable stimulus or aay from an unfavorable one, the

fre&uency of tumbles is lo, thus the cell moves toards or aay from the stimulus as

appropriate. Aoever, hen simming toards an unfavorable or aay from a favorable

stimulus, the fre&uency of tumbles increases, alloing the cell to reorient itself and move to

a more suitable groth.

liding motility is the movement of cells over surfaces ithout the aid of flagella, a trait

common to many bacteria, yet the mechanism of gliding motility is unknon. The glidingmotility apparatus hich propels the cells involves a comple% of proteins, yet the actual

nature of the motor and ho the components interact is not understood.

simple, differential staining and motility

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