Counting BacteriaCounting Bacteria

Serial DilutionsSerial Dilutions

Because of their very ______ ______, counting the number of bacteria in a sample can be _________

Although _______ _______ are possible with a ______________, they require a lot of time and ___________

Why Count the Number of Why Count the Number of Bacteria??Bacteria??

• ________ _____________:________ _____________:– How effective drugs are in killing How effective drugs are in killing

microorganisms?microorganisms?

• ___________________:___________________:– How many MO in CSF, blood, urineHow many MO in CSF, blood, urine

• ___________ ____________: - Contaminants in food, milk, water

Total Cell CountTotal Cell Count

• The most common method of The most common method of _______________ the total microbial _______________ the total microbial cells (______ ___ _________) cells (______ ___ _________)

• The ______ ___________ of cell The ______ ___________ of cell suspension in a __________ suspension in a __________ _____________ of known _______ _____________ of known _______ using a ________________ using a ________________

Total Cell Count – Both Total Cell Count – Both living and dead MOliving and dead MO

• Disadvantage: Disadvantage: ________________________________________________________________________________________________________________________________________________________________________________________________________________________

• Advantage: Advantage: __________________________________________________________________________________________________________________________________________________________________________________________________________________________

Total Cell Count – Both Total Cell Count – Both living and dead MOliving and dead MO

• Neubauer ___________ ___________Neubauer ___________ ___________

• Special _______ ____ _______ _______, Special _______ ____ _______ _______, microscopic marks on slide, known microscopic marks on slide, known __________________

•

Counting ChamberCounting Chamber

•Use special ______________ ____ Use special ______________ ____ __________________________________________

•Count specific areas of Count specific areas of __________________, multiply by __________________, multiply by __________________ ________________________________ ______________

Counting ChamberCounting Chamber

InstrumentationInstrumentation

• * * _____________________ (Light Meter)_____________________ (Light Meter)• Each single ____ stops some ______ from Each single ____ stops some ______ from

________________________________________________________________________________________• One side _____ ________, then test tube One side _____ ________, then test tube

containing MO, other side _______ containing MO, other side _______ ________________________________

• Needle reading from scale, then Needle reading from scale, then ________________ _____________ telling # of ________________ _____________ telling # of ____________________________________________________________________________________

Coulter CounterCoulter Counter

• Another method for _____________ Another method for _____________ __________________________

• An electronic instrument An electronic instrument “_____________ _________”“_____________ _________”

• Has a tiny tube (_____ ______ Has a tiny tube (_____ ______ _____________) that lets through _____________) that lets through only one cell at a timeonly one cell at a time

Viable Cell Count –Viable Cell Count – Living Cells OnlyLiving Cells Only

An easier method is to ______ ________ _____ __ _____ ______ (nutrient agar plate) and count the number of ________ _____ ______

If the bacteria are ______ ____ ______, each bacterial cell in the original sample should produce a _______ _______

Viable Count – Living Cells Viable Count – Living Cells onlyonly

Serial DilutionsSerial Dilutions

Bacterial samples must be _______ considerably to obtain ___________ ___________

To determine the ________ ___ ______ in a bacterial culture carry out _______ ____________

Serial DilutionsSerial Dilutions

Bacterial cell numbers are usually _______ _____ _____________________________

Plating out this sample, undiluted, would lead to the creation of a ___________________

Bacterial Lawn - many individual bacteria colonies that are ______________________ ____________________________________

Serial DilutionsSerial Dilutions

Bacterial cell numbers _____ __ __ _______

This is done by ______ ________ the amount of bacteria you have in your sample

A ______ _________ of bacteria sample is mixed with a _____ _______ (sterile broth or water), and then _______ ___________ are made

Serial DilutionsSerial Dilutions

A small amount of _____ ___ ____ _______ _________ ________ is then spread onto an ______ ________

The numbers of bacterial colonies that ______________________________

Serial DilutionsSerial Dilutions

Work ___________ using multiplication with the “___________ ____________”

________ ________ - the ______ ___ ______ that you have diluted the bacteria sample with the diluent solution

Make a determination of the numbers of bacteria in ______ ________ _________.

Serial DilutionSerial Dilution

Serial DilutionsSerial Dilutions

*Make ________ _____________*Make ________ _____________

*Spread a small amount from each tube *Spread a small amount from each tube onto _______ _______onto _______ _______

*Culture bacteria for a ________ __ *Culture bacteria for a ________ __ __________________

*Count colonies*Count colonies

________________________________________________________________

Membrane FiltrationMembrane Filtration

• ________ __________ can also be ________ __________ can also be used to determine the ________ used to determine the ________ ______________________________

• In this method ______ _____ In this method ______ _____ ___________ onto membrane filter ___________ onto membrane filter which is then placed over a which is then placed over a ________ ______ __________.________ ______ __________.

Membrane FiltrationMembrane Filtration

• The methodology is similar to The methodology is similar to conventional _______ ________ conventional _______ ________ _______________. _______________.

• Membranes have a printed Membranes have a printed millimeter ________ and colonies millimeter ________ and colonies can be counted under a _________ can be counted under a _________ _________________._________________.

Factors Factors essentialessential for __________ ________ for __________ ________

--proper ________________--proper ________________

--proper ___________________ (space, temperature)--proper ___________________ (space, temperature)

--No inhibiting factors--No inhibiting factors

Factors that _____________________ Factors that _____________________ ____________________________________________________________________________________

--lack of _____________--lack of _____________

--lack of __________ __________________--lack of __________ __________________

--presence of an ___________ __________--presence of an ___________ __________