EXPERIMENT 1:

References

Single cycle growth curves

1. Adams, M. H., Bacteriophages, Interscience Publishers, Inc., N. Y.,
1959, Appendix, p. 473 f£.

2. Epstein, R. H. et al., Physiological Studies of Conditional Lethal
Mutants of Bacteriophage T4D, Cold Spring Harbor Symposium 28, 375,

1963.

3. Stent, G. S., Molecular Biology of Bacterial Viruses, W. H. Freeman
and Co., San Francisco, 1963.

Bacteria

E. coli strains:

 

1. CR63 --
2. B a
3. K37 =
4. K3@ <--
5. ¢€3000--

Bacteriophage

amber-permissive, allowing growth of T4 amber mutants

amber non-permissive

amber-permissive, Hfr, allowing growth of MS2 amber mutants:
amber non-permissive, Hfr. Parent of K37

amber, non~permissive, Hfr

1. T4D and amber mutants

2. MS2 and amber mutants

Media

For MS2:

Bacto~tryptone 10 gm
Yeast extract 1 gm
Sodium chloride 8 gm

Glucose

1 gm

Calcium chloride 0.002 M
Distilled water 1 liter

For T4:
Bacto nutrient broth 8 gm
Bacto peptone 5 gm
Sodium chloride 5 gm
Glucose 1 gm

Distilled water 1 liter
-2-

The purpose of this experiment is to compare the growth curves of T4
and MS2 and "early" and "late" amber mutants of these phages. Time curves
of phage formation will be done in permissive hosts and infective centers
and 60 minute values only will be determined in non-permissive hosts.

Each pair of students will use a single phage type as follows:

Pair

T4r in CR63 and B

MS2 in K37 and K38

T4amNi22 in CR63 and B

MS2am9 in K37 and K38

T4amN120 in CR63 and B [7 6 Z }
MS2am2 in K37 and K38

Dunk wner
1

To be supplied:

1. CR63, B, K37, K38 at 2 x 108/ml
2. Phage suspensions at 2 x 108 pfu/ml
3. MS2 medium; T4 medium

Protocol for permissive hosts

-5' = Incubate 1 ml of host cells in 13 x 100 mm tubes at 37°
O' - Infect at moi of 0.1
5' + Dilute to 1072, 1073, 1074 with appropriate medium (T4 or MS2)

(All 1072 dilution steps should be done by transferring 0.1 mi to 10
ml, and all 1071 dilution steps by transferring 0.5 ml to 4.5 ml) Plate
0.1 mi of 1072, 1073, 10°4 dilutions immediately on permissive host.

 

Treat original culture with 1 drop of chloroform and continue incu
bating this tube (called the “original tube").

Continue incubating the 10°4 dilution only (called the "incubation
tube'').

10' - Remove 0.5 ml from the incubation tube into 13 x 100 mm tube.
Add 1 drop chloroform and shake.

15' - Repeat sampling as at 10'.

20' - Repeat sampling
Also plate 0.1 ml from original tube after diluting to 1072

and 1073.
25')
35')- Repeat sampling
45')

60' - Sample MS2 only

Handling of samples

oft 4
To va oe pion 0.1 ml lysozyme-EDTA and incubate at 37° for

30'.
-3-

T4 samples - incubate at 37° for 30'.
Plate 0.1 ml of following final dilutions, using permissive host (you
start with 1074):

10’, 15' - 1074, 1075

20' - 1075, 1076

25! - T4: 1075, 1076; ms2: 1075, 1076, 10-7
35', 45' = T4: 1075, 1076; MS2: 1076, 1077

60! - MS2: 1077, 107

Protocol for non-permissive hosts

Same as for permissive hosts up to 10'
Always plate on permissive host!
15' - Plate 0.1 ml from original tube after diluting to 10-2 and 1073
60' ~ Remove 1 ml from incubation tube. Add 1 drop of chloroform and
shake. u t mL °
For MS2¥7iftreat with chloroform and lysozyme-EDTA as described abo e and

plate 0.1 ml of 1074, 1075, 1076 dilutions MS Qz F& fEC4 TY

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