DIFFERENTIAL STAINING: THE ENDOSPORE STAIN

Vegetative cells are bacteria that are actively growing, metabolizing and dividing. When
vegetative cells are subjected to environmental stresses such as nutrient deprivation they
eventually die. However, some bacteria such as the Bacillus spp. and the Clostridium spp
can circumvent the problems associated with environmental stress by forming
endospores. Endospores are dorant or metabolically inactive forms of a bacterium
that allow it to survive the harsh environmental conditions. Spores are resistant to heat,
U radiation and chemicals because they are comprised of a tough proteinaceous
covering called !eratin. " mature endospore contains a complete set of the genetic
material #$%"& from the vegetative cell, ribosomes and specialized enzymes.
Endospores may be located in the iddle of the bacterium #central&, at the end of the
bacterium #terinal& and near t!e end of the bacteria #s"#terinal& and may be
spherical or elliptical. $at"re endospores are released from the vegetative cell to
become %ree endospores. When the free endospores are placed in an environment that
supports growth, the endospores will revert bac! to a vegetative cell in a process called
germination. 't should be noted that unli!e the process of binary fission observed with
vegetative cells, endospore formation is not a reproductive process but a process of
di%%erentiation that provides the bacteria with a mechanism for survival.
(ost endospore forming bacteria are found in soil or a)uatic environments.
However, some species of Bacillus and Clostridium have edical signi%icance.
Clostridium perfringens, C. botulinum and C. tetani are the causative agents of gas
gangrene, botulism and tetanus, respectively. Bacillus anthracis and Bacillus cereus are
the causative agents of anthra* and a self limiting food poisoning, respectively.
'n this e*ercise, you will perform an endospore stain on Bacillus spp. a
nonpathogenic, spore forming +ram positive rod. ,he instructor will provide a sample
containing bacteria that have been grown under different conditions- bacteria grown for
./0 hours, vegetative bacteria1 bacteria grown for 2./.0 hours, vegetative bacteria
containing endospores and bacteria grown for 32 hours1 mature free endospores.
" di%%erential staining techni)ue #the Schaeffer/4ulton method& is used to
distinguish between the vegetative cells and the endospores. " priar& stain
'alac!ite green( is "sed to stain t!e endospores. 5ecause endospores have a !eratin
covering and resist staining, the malachite green will be %orced into the endospores by
!eating. 'n this techni)ue heating acts as a ordant. )ater is "sed to decolori*e the
cells1 as the endospores are resistant to staining, the endospores are e)ually resistant to
de/staining and will retain the primary dye while the vegetative cells will lose the stain.
,he addition of a counterstain or secondary stain #safranin& is used to stain the
decolorized vegetative cells. When visualized under microscopy the cells should have
three characteristics- the vegetative cells should appear pin!, the vegetative cells that
contain endospores should stain pin! while the spores should be seen as green ellipses
within the cells. (ature, free endospores should not be associated with the vegetative
bacteria and should be seen as green ellipses.
6
T!e endospore stain:
6. 7repare a heat fi*ed smear on a clean microscope slide of the bacteria as shown by the
instructor. ,o this end the instructor will provide 8 plates with endospore producing
bacteria that have been grown on the plates for ./0 hours, 2./.0 hours and 32 hours.
7lace a small drop of sterile water on the slide and add a very small ali)uot of the bacteria
to the water drop with an inoculating needle. Use a sterile inoculating loop to ma!e a
very thin smear of the bacteria on the surface of the slide. "llow the smear to dry
completely.
2. 7lace a small piece of blotting paper over the smear and place the slide #smear side
up& on a staining rac! that has been placed over a boiling water bath in the corner of the
room.
8. 7ut on gloves and leave such gloves on until you finish staining the specimen.
.. 9ompletely cover the blotting paper that covers the smear with malachite green and
allow the slide to heat thoroughly over the water bath for 6: minutes. 5e certain to
replace any dye that has evaporated with fresh dye.
;. "fter 6: minutes carefully remove the slide from the rac! using a clothespin, tip off
any e*tra stain into a pan that is adjacent to the bath and ta!e the slide bac! to your
bench.
<. =emove the blotting paper and allow the slide to cool to room temperature for 2
minutes.
3. Use water to wash the malachite green from both sides of the microscope slide.
0. Stain the smear with safranin for 2 minutes.
>. =inse both side of the slide to remove the secondary stain and use the boo!let of
bibulous paper to blot the slide.
6:. Save your slide for the observation ne*t lab period.
O#serve t!e #acteria "nder +,,,- 'oil iersion( total agni%ication.
At t!is point &o" s!o"ld #e a#le to identi%& t!e vegetative cells/ t!e endospore
containing cells and t!e %ree endospores. Dra0 a pict"re o% 0!at &o" o#serve 0it!
icroscop& noting t!e color o% t!e cells and t!e spores.
2
Do t!e spores appear elliptical or sp!erical1
Are t!e spores central/ terinal or s"#terinal1
Ho0 an& spores are present in eac! #acill"s1
8