Question

EXPERIMENTAL PROTOCOL:

This exercise will engage students in examining uptake of green fluorescent-tagged bacteria by macrophage cells and unveil the endocytic mechanism used by them. Half of the class will assess bacterial endocytosis by macrophages at different time points, while the other half of the class will test the impact of a endocytosis-altering compound on bacterial uptake.

THIS TABLE OF CONTEXT BELOW IS THE GRAPH INFO ON ALL THE DATA COLECTED FROM THE STUDENTS. 1/2 STUDENTS USED THE CONTROL THE Latrunculin WITH THE E COLI. OTHER 1/2 CLASS DID IT WITHOUT THE CONTROL (MACROPAGES WITH E COLI.). IN STEP 9 EACH GROUP WAS ASSIGNED A DIFFERT TIME OF INCUBATION RANGING FROM 0,15,30 MIN.

different incubation time	15 min	0 min	30 min	15 min	30 min	30 min
Macrophages human cells	11.4 (group 5)	9.4 (group 1)	2.4 (group 4)	5.4 (group 11)	0 (group 10)	14.4(group12)
Latrunculin A stock solution DMSO	3.4 (group 6)	3.4 (group 7)	2.8 (group 9)	9.8 (group 3)	7.0 (group 2)
different incubation time	15 min	30 min	0 min	0 min	15 min

1. Summarize your interpretation of the findings.

2.  Include a statement about the future scientific directions suggested by your findings from the experiment (suggest future experiments using as base the results obtained in the lab)

4. Creat a graph with data given. A STANDARD DEVIATION AND AVERAGE graph.

Answer 1

Before I discuss the experimental analysis I would like to discuss the basic terms involved in this experiment.

ENDOCYTOSIS BY MACROPHAGES : Endocytosis is the process of uptake and engulfment of the bacteria or pathogen by the macrophage to render the body of the pathogen.

MACROPHAGES : Macrophage is a type of white blood cell that causes phagocytosis of anything that is foreign (often acting as an allergen or immunogen) to the body.

GREEN FLOURESCENT TAGGED BACTERIA : Bacteria are often tagged with proteins of certain molecular weight exhibiting flourescence of different wavelengths. Flourescence emission makes the detection of bacteria easy. The purpose of tagging in this experiment explains the easy detection of bacteria when engulfed by the macrophages.

LATRUNCULIN A : Latrunculin A a toxin isolated from the red sea sponge acts as a potent inhibitor of immunological phagocytosis by the phagocytic cells such as macrophages. Latrunculin A in this experiment is used to see the effect of the inhibitor on the phagocytosis.

The experiment included two different aspects. One included the effect of macropahges on the E.coli and hence indicated the phagocytosis rate while the other included the effect of the inhibitor Latrunculin A on the phagocytosis of the bacteria.

1) The data explains readings taken at three different incubation periods i.e., 0min , 15min and 30min.    

Macrophages with endocytosed bacteria

0 min	15 min	30 min
9.4	11.4	24
0	5.4	14.4

   As the incubation period increased the number of macrophages that phagocytosed the bacteria increased as the flourescence increased with the incubation time.

Phagocytosis study with the inhibitor

0 min	15 min	30 min
28	3.4	3.4
9.8	7.0	Data Not Given

When the inhibitor of phagocytosis was used the rate of phagocytosis decreased with incubation time which is explained by the decreasing flourescence. The formation of the immune complex ( bacteria-antibody) occurs but the internalization of bacteria attached with the antibody into the macrophage does not occur.

2) Studies can be done where the effect of latrunculin A on the normal and activated macrophages can be compared. Phagocytosis includes the crucial element of actin polymerization. Actin polymerization is known to blocked by the presence of latrunculin A, so the effect of latrunculin A on the actin filaments during the course of incubation can be studied.

4) Average table is as follows :

The graphs showing Average and standard deviation for the macrophages with E.coli and for the use of inhibitor are as follows :

Bars outlined by blue indicates the average and the bars outlined by orange indicates the standard deviation.