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Culturing Microorganisms > Counting Bacteria
Counting Bacteria
• Direct Counting
• Viable Cell Counting
• Measurements of Microbial Mass
• Detecting Acid and Gas Production
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Culturing Microorganisms > Counting Bacteria
Direct Counting
• Directly counting blood cells or tissue cells by using a hemocytometer can
determine the concentration of a known volume.
• Counting the number of colonies that arise on a pour plate can calculate the
concentration by multiplying the count by the volume spread on the pour plate.
• Direct counting methods are easy to perform and do not require highly specialized
equipment, but are often slower than other methods.
Counting Colonies
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Culturing Microorganisms > Counting Bacteria
Viable Cell Counting
• The spread plate relies on bacteria growing a colony on a nutrient medium so that
the colony becomes visible to the naked eye and the number of colonies on a
plate can be counted.
• Selective media can be used to restrict the growth of non-target bacteria.
• The pour plate method is used when the analysis is looking for bacterial species
that grow poorly in air, for example water samples.
Selective media can be used to restrict the
growth of non-target bacteria.
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Culturing Microorganisms > Counting Bacteria
Measurements of Microbial Mass
• Calculating the dry weight of a sample enables one to calculate the cell count, but
the sensitivity is limited to samples containing more than 10E8 bacteria per
milliliter.
• Spectrophotometry is an indirect method for calculating cell concentrations by
measuring the changes in turbidity.
• Bacteria can also be counted by using the plating method, which is based on the
number of colonies formed in Petri dishes containing specific growth media.
Spectrophotometer
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Culturing Microorganisms > Counting Bacteria
Detecting Acid and Gas Production
• Differential media uses the biochemical characteristics of a microorganism
growing in the presence of specific nutrients or indicators.
• To measure acid production one can use a pH indicator in the media.
• The Durham tube method is used to detect production of gas by microorganisms.
Using differential media to detect acid production
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Appendix
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Culturing Microorganisms
Key terms
• differential media Differential media or indicator media distinguish one microorganism type from another growing on the same
media.
• flow cytometry A technique used to sort and classify cells by using fluorescent markers on their surface.
• gravimeter An instrument used to measure local variations in the gravitational field
• hemocytometer A device that counts microscopic particles. The hemocytometer works by creating a volumetric grid divided into
differently sized cubes for accurately counting the number of particles in a cube and calculating the concentration of the entire
sample.
• plate count A means to identify the number of actively growing cells in a sample.
• spectrophotometry A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions.
However they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 200nm
- 2500nm using different controls and calibrations. [2] Within these ranges of light, calibrations are needed on the machine
using standards that vary in type depending on the wavelength of the photometric determination. [3]
• streak plate A petri dish with a growth medium.
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Culturing Microorganisms
Using differential media to detect acid production
Sorbitol fermenting non pathogenic commensal bacteria from faeces growing on Cefixime Tellurite Sorbitol MacConkey Agar.
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Flickr. "All%20sizes%20%7C%20Cefixime%20Tellurite%20Sorbitol%20MacConkey%20Agar%20(CT-SMAC%20Agar)%20%7C%20Flickr%20%20Photo%20Sharing!." CC BY http://www.flickr.com/photos/nathanreading/6141227071/sizes/z/in/photostream/ View on Boundless.com
Culturing Microorganisms
Selective media can be used to restrict the growth of non-target bacteria.
Urine cultured on Oxoid Brilliance UTI Agar plate. 1uL of urine spread onto the agar surface. The top sample is from patient with clinical urinary tract
infection (UTI). The bottom sample is a mixed culture.
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Culturing Microorganisms
Counting Colonies
An example of counting colonies on a streak plate.
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Wikimedia. "EPA GULF BREEZE LABORATORY, THE MICRO-BIOLOGY LAB.TAKING A BACTERIA COLONY COUNT - NARA - 546274." Public domain
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Culturing Microorganisms
Spectrophotometer
This spectrophotometer can measure as little as one microliter of a sample.
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Culturing Microorganisms
Aliens come to earth and find a microbiology lab. They find a
device which has a microscope set of grids or cubes on it. What
did they find?
A) counting cube
B) microscope
C) volumetric flask
D) hemocytometer
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Culturing Microorganisms
Aliens come to earth and find a microbiology lab. They find a
device which has a microscope set of grids or cubes on it. What
did they find?
A) counting cube
B) microscope
C) volumetric flask
D) hemocytometer
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Culturing Microorganisms
You have a powerful microscope and lots of time. You take a
bacteria culture and count the number bacteria in it, however you
don't know the total viable number, why?
A) some bacteria are too small to be counted
B) you lost count
C) can't determine if cells are viable or not
D) could be dirt in the culture
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Culturing Microorganisms
You have a powerful microscope and lots of time. You take a
bacteria culture and count the number bacteria in it, however you
don't know the total viable number, why?
A) some bacteria are too small to be counted
B) you lost count
C) can't determine if cells are viable or not
D) could be dirt in the culture
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Culturing Microorganisms
Which of the following are ways of measuring microbial mass?
A) spectrophotometry
B) All of these answers
C) gravimeter method
D) direct counting
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Culturing Microorganisms
Which of the following are ways of measuring microbial mass?
A) spectrophotometry
B) All of these answers
C) gravimeter method
D) direct counting
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Culturing Microorganisms
Sorbital MacConkey Agar can be used to detect changes in
______________?
A) termperature
B) pH
C) gas content
D) eosin methylene blue
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Culturing Microorganisms
Sorbital MacConkey Agar can be used to detect changes in
______________?
A) termperature
B) pH
C) gas content
D) eosin methylene blue
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Culturing Microorganisms
Attribution
• Wikipedia. "Bacteriological water analysis." CC BY-SA 3.0 http://en.wikipedia.org/wiki/Bacteriological_water_analysis
• Wikidot. "Serial Dilutions/counting viable bacteria in a liquid sample - Microbiology@Morningside." CC BY
http://morningsidemicro.wikidot.com/serial-dilutions
• Wikidot. "Ways To Count Bacteria - Microbiology@Morningside." CC BY-SA http://morningsidemicro.wikidot.com/ways-tocount-bacteria
• Wikipedia. "plate count." CC BY-SA 3.0 http://en.wikipedia.org/wiki/plate%20count
• Wikidot. "Ways Of Counting Bacteria - Microbiology@Morningside." CC BY-SA http://morningsidemicro.wikidot.com/ways-ofcounting-bacteria
• Wikidot. "Ways Of Counting Bacteria - Microbiology@Morningside." CC BY-SA http://morningsidemicro.wikidot.com/ways-ofcounting-bacteria
• African Virtual University. "Microbiology and Mycology." CC BY-SA http://oer.avu.org/handle/123456789/36
• http://wiki.biomine.skelleftea.se/wiki/index.php/Microbial_growth. "Microbial growth - BioMineWiki." CC BY
http://wiki.biomine.skelleftea.se/wiki/index.php/Microbial_growth
• University of South Carolina. CC BY http://pathmicro.med.sc.edu/2010bactpdf/26%2520Nutrition%2520growth%2520and%2520metabolism%25202010.pdf
• Wikipedia. "Microbiological culture." CC BY-SA 3.0 http://en.wikipedia.org/wiki/Microbiological_culture
• Boundless Learning. "Boundless." CC BY-SA 3.0 http://www.boundless.com//microbiology/definition/streak-plate
• Boundless Learning. "Boundless." CC BY-SA 3.0 http://www.boundless.com//microbiology/definition/hemocytometer
• African Virtual University. "Microbiology and Mycology." CC BY-SA http://oer.avu.org/handle/123456789/36
• Wikibooks. "NZ Level 6: Experimental sciences/Life processes." CC BY-SA 3.0
http://en.wikibooks.org/wiki/NZ_Level_6:_Experimental_sciences/Life_processes#Culturing_Microorganisms
• Wikipedia. "Cell counting." CC BY-SA 3.0 http://en.wikipedia.org/wiki/Cell_counting
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• Wikipedia. "Bacterial growth." CC BY-SA 3.0 http://en.wikipedia.org/wiki/Bacterial_growth
Culturing Microorganisms
• University of South Carolina. "Bacterial nutrition, growth and energy metabolism." CC BY
http://pathmicro.med.sc.edu/fox/nutrition.htm
• Wikipedia. "Cell counting." CC BY-SA 3.0 http://en.wikipedia.org/wiki/Cell_counting
• Wikipedia. "Cell counting." CC BY-SA 3.0 http://en.wikipedia.org/wiki/Cell_counting
• Wikipedia. "Bacteria." CC BY-SA 3.0 http://en.wikipedia.org/wiki/Bacteria#Growth_and_reproduction
• Wiktionary. "flow cytometry." CC BY-SA 3.0 http://en.wiktionary.org/wiki/flow+cytometry
• Wiktionary. "gravimeter." CC BY-SA 3.0 http://en.wiktionary.org/wiki/gravimeter
• Wikipedia. "spectrophotometry." CC BY-SA 3.0 http://en.wikipedia.org/wiki/spectrophotometry
• Wikipedia. "Growth medium." CC BY-SA 3.0 http://en.wikipedia.org/wiki/Growth_medium#Differential_media
• Wikipedia. "Durham tube." CC BY-SA 3.0 http://en.wikipedia.org/wiki/Durham_tube
• Wikipedia. "Coliform bacteria." CC BY-SA 3.0 http://en.wikipedia.org/wiki/Coliform_bacteria
• Wikipedia. "Bacteria." CC BY-SA 3.0 http://en.wikipedia.org/wiki/Bacteria#Growth_and_reproduction
• Wikipedia. "Microbiology." CC BY-SA 3.0 http://en.wikipedia.org/wiki/Microbiology
• Wikipedia. "Sorbitol-MacConkey agar." CC BY-SA 3.0 http://en.wikipedia.org/wiki/Sorbitol-MacConkey_agar
• Wikipedia. "Microbiological culture." CC BY-SA 3.0 http://en.wikipedia.org/wiki/Microbiological_culture
• Boundless Learning. "Boundless." CC BY-SA 3.0 http://www.boundless.com//microbiology/definition/differential-media
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