Wednesday, May 6, 2009

An apparently unnecessary step

I love old papers. It’s true that they’re often long and obscure, but they also asked questions at a more fundamental level than papers today and with significantly less jargon. My favorite example is this early paper by Seymour Benzer, in which he does things like invent the complementation test and show the likely linear nature of the genetic material.

I’ve been slowly working my way through another old paper, this one on transformation in Haemophilus influenzae Rd:

GOODGAL and HERRIOTT. Studies on transformations of Hemophilus influenzae. I. Competence. J Gen Physiol (1961) vol. 44 pp. 1201-27

This work established many important facts about Haemophilus transformation that we luckily can take for granted today. Later, I’ll undoubtedly touch on other aspects of this paper (namely their measurements of co-transformation and calculations of how many cells in a culture are competent), but right now, I just want to point out a bit of the methods. As I read this section, I was at first incredulous:
Preparation of Levinthal Stock for Growth of H. influenzae (1): To a 6 liter flask add 2100 ml distilled H~O and 74 gm Difco dehydrated brain heart infusion. Bring this solution to a vigorous boil, remove from heat, and add cautiously 200 ml defibrinated sheep's blood. The defibrinated sheep's blood is obtained by stirring fresh blood with a rough wooden paddle immediately after it is taken from the animal. Stirring vigorously for 5 minutes with a motor-driven paddle or for 20 minutes by hand should be sufficient. The blood is strained through cheese-cloth and stored frozen before use. Medium made from unfrozen blood is usually turbid. The first addition of blood caused a heavy evolution of gas from the broth producing a violent foaming. Therefore, only 5 ml of blood should be added initially to the hot infusion, and no more added until the gas evolution ceases. Continue to add as rapidly as foaming will permit, until the entire 200 ml is introduced...

The protocol continues on, but when I reached footnote (1), indicated in the heading, I actually laughed aloud on the bus the other day (emphasis mine):

(1) Since this work was completed, Mr. John Cameron and Mr. Harold Isaacson working in this laboratory have found that growth and development of competence of H. influenzae equal to that obtained with Elev broth were observed in a 3.5 per cent solution of Difco brain heart infusion mixed 3:1 with 3 per cent Eugonbroth appropriately supplemented with 10 pg/ml of hemin and 2 /~g/ml of DPN. This medium has been used with uniform success for a number of months. It eliminates a time-consuming and apparently unnecessary step of the addition of fresh blood.
Science marches on (and gets easier). I am thankful to Mr. Cameron and Mr. Isaacson for making my life significantly less disgusting. (Unfortunately for me, their methods do report 5-10% transformation frequencies... If these are to be believed, I might need to try using Levinthal Stock for some of my more transformation rate sensitive experiments. Alas.)

1 comment:

  1. My favourite old paper is the first one to consider applying concepts of topology to DNA; I think it's by Sydney Brenner and Francis Crick. It starts with the wonderful statement that "DNA is such an important molecule that it's impossible to know too much about it."