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Microbiology Practical 1

1a. What is Autoclave used for?

It is used for the liquid substances, Medias and clinical equipments. It is also called the wet

heat sterilization. The temperature used is 121 ! for 2" to #" minutes. $ simple %ersion of$utocla%e is pressure coo&er. In $utocla%e, steam pressure is built to a 1' lbs per square inchat 121 ! for about 2" to #" minutes.

1b. Mention the temperature and pressure achieved for sterilization of laboratorymaterials and hospital supplies . P.(2 I) !*+* I)-

$utocla%es commonly use steam heated to 121 1#( ! /2'" 20# .

!ontrol of microorganisms is critical in a laboratory or hospital setting. Two general types ofagents are a%ailable for limiting microbial populations3

1. Physical agents3 heat, filters, and radiation

2. !hemical agents3 antiseptics, germicides, antibiotics, and disinfectants

To ensure the autocla%ing process was able to cause sterilization, most autocla%es ha%emeters and chart that record or display pertinent information such as temperature and

pressure as a function of time. $utocla%es commonly use steam heated to 121 1#( ! /2'" 20# . To achie%e sterility, a holding time of at least 1' minutes at 121 ! /2'" or #minutes at 1#( ! /20# is required. $dditional sterilizing time is usually required for

liquids and instruments pac&ed in layers of cloth, as they may ta&e longer to reach therequired temperature /unnecessary in machines that grind the contents prior to sterilization .

1c. Name the bacterium used for quality control of sterilization

4terilization is the process of destroying all manner of li%ing things, including bacterialspores, the most resistant to destruction of all life forms.

5acillus stearothermophilus considered ideal for monitoring because this organism lac&s pathogenicity, pyrogenicity and to6icity. The number of spores that should be present whensterilization is being monitored is 1" ( to 1" 7 for 5.stearothermophilusand around 1" 7 for 5acillus subtilis %ar niger.

2a. Mention the temperature and duration to be used for sterilization hot air oven. p.(2and img 1(("

17" ! for 2 hrs or 7 to 12 min for 18" !. It cannot be used for sterilization culture media,alcoholic or other %olatile solutions.

2b. What are materials of clinical and laboratory importance that can be sterilized in

the hot air oven. p. 42

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*f the gram positi%es, 2 are cocci, and the other ( are rod9shaped/bacilli .

1. 4treptococcus and staphylococcus

2. orm spores3 5acillus and clostridium

#. )o spore formation3 !orynebacterium and +isteria

*f the gram negati%e organisms3 )eisseria it loo&s li&e 2 coffee beans &issing

The gram positi%e cell wall is %ery thic& and has e6tensi%e cross9lin&ing of the amino acidside chains. The gram positi%e cell en%elope has an outer cell wall composed of comple6cross9lin&ed peptidoglycan, teichoic acid, polysaccharides, and other proteins. The innersurface of the cell wall touches the cytoplasmic membrane. The cytoplasmic membrane

contains proteins that span the lipid bilayer. The bacterial cytoplasmic membrane/unli&e thatof animals has no cholesterol or other sterols. $n important polysaccharide present in thegrams positi%e cell wall is teichoic acid. It acts as an antigenic determinant, so it is importantfor serologic identification of many gram9positi%e species.

The gram negati%e cell wall is %ery thin with a fairly simple cross9lin&ing pattern.

Mycoplasma donAt ha%e a cell wall.

).'escribe the procedure for ma(in! a bacterial smear for microscopic observation* don+t for!et aspectic technique,.

There are ( steps to the gram stain3

1.Pour on crystal %iolet stain/a blue dye and wait 7" seconds.

2.;ash off with water and flood with iodine solution. ;ait 7" seconds.

#. ;ash off with water and then <decolorize= with 8'> alcohol.

(. inally, counter9stain with safranin/ a red dye . ;ait #" seconds and wash off withh2o.

!ells that absorb the crystal %iolet and hold onto it will appear 5+?@ and are calledgram positi%e.

If the crystal %iolet is washed off by the alcohol, these cells will absorb the safraninand appear red and are called gram negati%e.

a. Why should a bacterial smear be air-dried before heat fi in! */flamin!0, it?

To allow the e6cess water to dry out before heating. If heated right after, the water wouldcause the smear to o%erheat and denature some features in the stain and those featureswould no longer be seen.

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