Culturing, Counting, and identifying microbes in ecosystems (CH 26)

what is axenic?

a pure culture

What is a possible solution for culturing microbes with fastidious growth requirements?

• assess growth capabilities of similar microbes

• grow in diffusion chambers

What are 2 possible solutions for culturing microbes that rely on signals or cross-feeding from other microbes?

• co-cultivate

• use diffusion chambers

Are all bacteria culturable? If not, what are some potential reasons why?

• no

• fastidious growth requirements

• dependence on other communities

• dormant or in a viable but not culturable state

What does the great plate count anomaly refer to?

number in a sample is significantly higher than the number able to be cultured on a plate

Describe 1:10 serial dilution and what it is useful for

• used to estimate microbial population sizes in a sample

• best way to get viable count (if bacteria is culturable)

• dilute 1ml of sample to 9mL of broth, taking 1mL from each successive sample and diluting in 9mL of broth

• streak 3 plates for each broth, and find countable plates with 30-300 colonies

• Calculate the number of colonies/mL in sample

  • ex. if 1/10,000 dilution with 32 colonies, 32×10,000 = 320,000 bacteria/mL in sample

If a 1:10 serial dilution has been performed 3 times and 122 distinct colonies are formed, how many bacteria are present per mL of sample?

• 10³ = 122 x 10^3 = 122,000 bacteria/mL in sample.

If a 1:10 serial dilution are on a plate of 1/100,000 dilution and 33 distinct colonies are formed, how many bacteria are present per mL of sample?

• 100,000×33 = 3,300,000 bacteria/mL in sample.

What is a urinalysis plate count?

• used to measure bacteria in urine sample

• use .001/.0001mL loops

• draws a line down the middle of a TSA plate then cross streaks

• must have greater than 10^5 microbes/mL to be clinically relevant

How does a Most probable number (MPN) estimate work?

1. serially dilute sample in growth broth several times

2. create 3 1mL tubes from each dilution

3. count number of tubes with growth

4. use a standardized MPN table to estimate bacteria/mL

What are coliforms? Include their morphology, fermentation, and significance.

• gram negative bacilli

• non spore forming

• ferment lactose to acid and gas

• present in feces of warm-blooded animals, wide spread in soil

• indicator organisms of potential fecal contamination

How are coliforms in water often initially counted?

using MPN, dilute multiple times and inoculate several tubes from each dilution. infer concentration using standardized MPN table using number of positive tubes

What are the steps for confirming coliforms in water? Include the counting method and decisiveness of each test

1. presumptibe test: MPN on samples, dilute multiple times, inoculate multiple tubes from each dilution and refer to standardized MPN table for bacteria/mL based on number of positive tubes

2. confirmatory test: 90% of E. coli strains have GUD. from each MPN tube with positive growth, test for presence of GUD cleaving MUG to MU. will be fluorescent.

3. complete test: plate positives from confirmatory test on EMB agar. selects against gram positive bacteria and E. coli will appear metallic green. confirms fecal contamination

What are other ways to tell viable cells other than culturing?

• staining

• FAME analysis

• MALDI-ToF

• sequencing

What is FAME analysis and what are some limitations?

• fatty acid methyl ester

• fatty acids from cell membranes extracted and looked at for chain length, degree of saturation, branching, and hydroxyl groups

• microbes of the same species grown under specific conditions have identical FAME profiles

• limitations: must be able to grow in pure culture

What is mass spectrometry MALDI-ToF? What are the advantages and limitations?

• matrix-assisted laser desorption/ionization time of flight

• generates a specific protein profile to ID microbe

• advantage: fast, affordable, accurate, good in clinical settings

• limitations: need pure culture, can’t identify new microbes

How can organisms be ID’d based on molecular techniques?

• signature sequences: sequence using PCR; can ID nonculturable organisms

• Whole genome sequencing: determine nucleotide identity to determine species

What are the methods of microbial community analysis?

• FISH - fluorescently tag microbes

• Metagenomics - comprehensive population diversity from DNA sequencing