First some background: The standard procedure is to supplement BHI with hemin and NAD from stock supplies that are already made up to standard concentrations (giving sBHI), to put 5ml of sBHI into a 25ml glass culture tube, to add H. influenzae cells either from a frozen stock or a previous culture (usually a 1/100 dilution), and to incubate the culture overnight on a roller wheel in the 37°C incubator. H. influenzae will normally also grow in the E. coli medium LB, provided it has been supplemented with hemin and NAD (sLB). E. coli doesn't need hemin or NAD and will grow well in both LB and BHI, better in BHI because it's a richer (and more expensive) medium. Growth can be checked various ways. The easiest is simply looking at the turbidity of the culture, but we can also measure the turbidity in a spectrophotometer, dilute the culture and plate the cells on agar to count the colonies, or look at the cells under a microscope. The last is not very useful for H. influenzae, mainly because the cells are so tiny but partly because our very expensive microscope is badly out of alignment. (We are arranging for a long-overdue visit from the serviceman.)
The first tests, done with the original problematic batch of BHI, found that inoculation of sBHI culture tubes with H. influenzae produced no growth but inoculation of E. coli directly into the bottles of BHI produced abundant growth.
The lab assistant then made four bottles of new BHI using combinations of the two stocks of BHI powder (old Difco brand and new MBL brand) and two sources of distilled water (secondary carboy and source carboy); this gave four bottles, labeled A, B, C, and D. One of the post-docs supervised this to make sure she wasn't making any errors. The post-docs then inoculated tubes of these media with H. influenzae from a freezer stock (amounts not carefully controlled, and with E. coli from a fresh culture. All of the H. influenzae tubes grew but none of the E. coli ones did! They then inoculated H. influenzae from the tubes that had grown into fresh tubes of media A-D, and this time the cells didn't grow!
I wanted to find out what the medium was doing to the cells by looking at them under the microscope. So yesterday I inoculated tubes of sBHI (from bottle A) and sLB with measured amounts of H. influenzae and of E. coli. Both inocula were prepared by resuspending cells taken from fresh colonies on plates into a small amount of LB, and then adding 50 microliters to 2.5ml of medium. I used disposable plastic culture tubes rather than our standard glass culture tubes because we wanted to exclude the possibility that dirty tubes were causing the problem.
I looked at the cells immediately after inoculation and after 30', 60' and 120' in the incubator. The E. coli cells in both sLB and sBHI did what healthy cells do - they gradually became longer and divided so that, after 120' the culture was very cloudy and each microscope 'field of view' contained 5-10 times more cells than it had at the start. The H. influenzae cells in sLB also grew. Because they're so little they looked like tiny specks and threads, but the number and proportion of threads got higher, indicating that the cells were elongating and dividing into new cells. But the cells in sBHI just sat there, continuing to look like a mixture of specks and short threads. The post-doc measured the culture turbidities in the spectrophotometer, confirming that E. coli was at high density in both media (higher density in the sBHI) and that H. influenzae was at higher density in sLB than in sBHI.
What did I learn? First, the problem is reproducible. Second, it isn't dirty culture tubes. Third, the problem manifests itself quite quickly. It isn't that the cells grow initially and then run out of some key nutrient - rather they don't grow at all. Fourth, the problem isn't the hemin or NAD. These results reinforce my notion that we should focus on the inability of H. influenzae to grow in BHI medium that does allow growth of E. coli, and not worry for now about the time when H. influenzae did grow and E. coli didn't.
So what will I do today? The post-doc made a big batch of BHI and BHI agar yesterday for me to do tests with, and streaked out H. influenzae and E. coli on agar plates.
- I think I'll first repeat yesterday's experiment with media A-D and the new batch (call it E), this time measuring the turbidities of all cultures at the start as well as after 2 hours.
- I'll also use oil-immersion to look at the H. influenzae cells under the microscope - this is a bit more hassle but gives higher resolution.
- I'll also dilute and plate the H. influenzae cells that are in the medium they wouldn't grow in yesterday. By doing this I can find out whether the cells die (and how quickly) or just fail to grow. Finding that the cells die would suggest that the medium contains something toxic, whereas finding that they just fail to grow would suggest that the medium is lacking an important nutrient.
- I'll also try mixing the sBHI 50:50 with LB. This might also show whether there's something missing from the BHI (if H. influenzae grows in the mixture) or something toxic in the BHI (if H. influenzae doesn't grow in the mixture).
- The batch E BHI agar was made with the same medium as batch E broth, so I'll pour plates of this and see if cells grow into colonies overnight. So far the problem has been found only with cells in liquid culture, but the liquid medium and agar have been from batches made on different days. (I can do my other plating on another batch of plates that one of the post-docs poured on Monday - we know cells do grow into colonies on these plates.)