Counting bacterial cells by using a drop plating method

Serial Dilutions

1.     Pipette 0.1 ml of bacterial suspension into a dilution tube containing 0.9 ml of sterile buffered water.

2.     Recap tube and vortex the tube for approximately 8 seconds.

3.     Using a new tip, pipette 0.1 ml of vortexed dilution tube into a second dilution tube containing 0.9 ml of sterile buffered water.

4.     Repeat process until there are four serial dilutions of the original 0.9 ml sample. 

Drop Plating

1.     Label the bottom of the LB agar plates by dividing them into fourths with a ruler and marking pen. Each serial dilution of the sample will occupy one quadrant of each plate.  Prepare plates in duplicate.

2.     Set the automatic pipetteman to pull up 100 µl of sample and expel 10 µl with each push of the button (slow setting).

3.     Vortex sample for approximately 8 seconds.

4.     Pick up sample with pippetteman.  Expel sample in five evenly spaced 10 µl drops onto the quadrant of one of the petri plates that have been labeled for that particular dilution of the sample. 

5.     Repeat this procedure with the duplicate plate.  Note:  If only a dedicated 10 ml pipette is available, 5 drops of 10 ml each should be placed on the plate.

6.     Vortex sample again for approximately 8 seconds.

7.     Pick up 100 µl of the next dilution and repeat steps 4-6.

8.     Repeat steps 4-6 for each tube in the dilution series.

9.     Let the drops soak into the media before turning plates over for incubation.

10.   Incubate overnight at 35-37˚C.

11.   Remove plates when colonies have developed and count the dilution which contains 3-30 colonies per 10 µl drop.  Viable cell counts are expressed as colony forming units (CFU)/surface area.

Example Calculation

Calculate LOG10 CFU/cm2 according to the following formula:

LOG (CFU/cm2) = LOG [(average CFU/drop volume)(dilution counted)(volume scraped into/surface area*)]  *scraped slide

= LOG [(average of plate counts/ 0.01 ml)(10dilution)(10 ml/1.267cm2)]

For example, if the average units per drop was 16.6 and they were counted in the third dilution, then the LOG (CFU/cm2) = LOG [(16.6 / 0.01)(103)(10/1.267)] = 7.12

Illustrations:

Figure 1. Dilution series followed by drop-plating technique for the Standard Method.

Turbidity measurement in log phase for Enumeration of Stenotrophomonas maltophilia D457

Using the spectrophotometer to estimating the number of bacteria in a liquid medium (we used LB broth) is to measure the turbidity or cloudiness of a culture and translate this measurement into cell numbers.  determine the optical density (O.D.) of the assigned broth culture at 600 nm. for Stenotrophomonas maltophilia D457

Stenotrophomonas maltophilia  (ATCC® BAA-84™)
Strain Designations     D457
Isolation: Bronchial aspirate, Madrid, Spain
Biosafety Level 2

The bacteria inoculated on LB agar to obtained isolated colonies, a single colony inoculated into 9 tubes each one contains 3 ml of LB broth, the tubes were kept in 37 C^၀ shaker incubator for 9 different period of time as fallowing: 6:30, 7:30, 9, 10, 14, 16, 17:30, 19 hours respectively; at the end of each period of time the OD600 of the broth culture was measured also a 8 tenfold dilution of the broth were made(1/10,1/100,1/1000……1/10^{^8}) Transfer 0.1 ml of the final dilution to triplicate LB agar plates.

Turb

Result:

CFU calculated using the formula  CFU= (average # of colonies x d.f)/volume of culture

Time in hours	CFU/ml	OD600
6:30	4.0*10^7	0.088
7:30	1.8* 10^7	0.217
9	2*10^8	0.4721
10	2.2*10^6	0.6048
14	4.1*10^8	0.9485
16	7.0*10^8	1.0971
17:30	1.9*10^8	0.8223
19	5.0*10^8	1.2402

 A concentration of 1.0* 10^7 per 40 micro liter = 2.5* 10^8  

Effect of live bacteria and bacterial supernatant on A549 cells attachment (3 hours incubation)in a 24 well plates

Same procedures describes in in the experiment " Effect of live bacteria on A549 cells attachment (2 hours incubation) in a 96 well plates" but using a 24 well plates seeded with A549 cells and adding 1 mL of bacterial broth or bacterial supernatant to each well with 1 mL of RPMI media . after 3 hours incubation time , the attachment of the cells were measured suing MTT assay.