UNIVERSITY OF CAPE TOWN
MOLECULAR AND CELL BIOLOGY DEPARTMENT
2019
COURSE:
MCB2023S
STUDENT PAIR DETAILS:
PRACTICAL DAY
BAY
NAME
STUDENT #
Thursday
17
Grace Nadin
NDNGRA002
PRACTICAL
5:
Genotype and Phenotype Exp 1 & 2
SUBMISSION DATE: 04 October 2019
DEMONSTRATOR:
ACADEMIC:
Dr Suhail Rafudeen
EXPERIMENT 1 & 2
Exp 1 Results Part A
MARK ALLOCATION
1
Exp 1 Results Part B
6
Question a
1
Question b
2
Question c
2
Exp 2 Results
27
Genotype
6
Question 1
2
Question 2
3
Question 3
1
Question 4
1
TOTAL
52
PERCENTAGE
MARK
Part A:
16 colonies grew on the NAL plate in a gradient; the greatest number were closest to 0µg/mL, with
far less towards the greatest concentration of NAL. There were 13 colonies in first half of plate, 3
colonies between halfway and ¾ and one in the last quarter.
Part B:
Dilution:
Number of colonies:
CFU/mL
10^-3
TMTC
-
10^-4
TMTC
-
10^-5
TNTC
-
10^-6
328
CFU/mL =
328 x 10^6
0.1
= 3.28 x 10^9
a. What fraction (%) of the bacterial culture was Nal resistant (calculate this from the results of Part
1A and Part 1B)?
CFU/mL on the NAL plate = 16 x 0.1mL = 160 CFU/mL
CFU/mL on NA plate = 3.28 x 10^9 CFU/mL
%NAL resistant = 100 x
160
t = 4.88 x 10^-6 %
3.28 x 10^9
b. How does Naladixic acid act as an antibiotic agent against bacteria?
Naladixic acid acts as an inhibitor of subunits of DNA gyrase and topoisomerase, enzymes involved in
bacterial DNA replication. Thus, nalidixic acid sensitive bacterium cannot replicate their DNA and
thus cannot grow or reproduce.
c. How do bacteria become resistant to Naladixic acid?
Bacteria become resistant to nalidixic acid by mutations. This could be a mutation that alters the
target enzymes or that affects the ability of naladicix acid to enter the cell. These mutations can be
transferred between bacteria by horizontal gene transfer. Bacteria with nalidixic acid resistance are
selected for.
Experiment 2: + growth ; - no growth
Strain:
Growth
on A
CSH63
Ura-, strep-, trp+,
his+, pro+
Lacz-, pro-, his-,
+
+
+
+
trp-, his-, ura-,
strep+
Lacz-, pro-, ura-,
+
+
+
+
+
+
trp+, his+, strep+
Ura-, strep-, trp+,
+
+
+
+
+
+
+
+
his+, pro+
Lacz-, pro-, ura-,
+
+
+
+
+
+
trp+, his+, strep+
Ura-, strep-, trp+,
+
+
+
+
+
+
+
+
his+, pro+
NOTE: there was not lactose on the MacConkey agar plates. For this reason, we cannot determine
from those plates if the bacteria were able to digest lactose or not.
CSH54
CSH50
CSH23
CSH56
CSH36
+
Growth
on B
-
Growth
on C
+
Growth
on D
+
Growth
on E
+
Growth
on F
+
Growth
on G
+
Growth
on H
Growth
on J
+
Genotype
+
(proline=pro, tryptophan=trp, histidine=his, lactose = lacz and streptomycin=strep)
1. A genotype is the genetic coding of genes within the genome of an organism whilst the
phenotype is the physical manifestation of those genes.
2. MacConkey agar is a selective-differential media. It selects for gram negative bacteria and
allows for differentiation between the bacteria that can metabolise lactose and those that
can’t. The media turns from pink to red when lactose is digested.
3. If a bacterium showed no growth on the MacConkey plate, it is likely because that bacterium
is gram positive. The growth of gram-positive bacterium is repressed by the crystal violet dye
in the medium.
4. The C-H plates are on a minimal media which means they must make most of their amino
acids, slowing their growth, whilst the A,B,J plates are on complex medias which provide
most of the nutrients required.