Blood and Tears Practical report

Introduction
The conjunctiva (or external surface of the eye) and the upper gastrointestinal tract are frequently exposed to a number of microorganisms every day. The conjunctiva can be exposed to harmful bacteria through hand to eye contact or colonisation of nearby nasal or sinus mucosal tissues. The eyes and mouth have defence mechanisms against bacteria. Contained within tears and saliva are 4 proteins known as lysozyme, lactoferrin, secretory IgA and lipocalin (Zhou, et al. 2012)(Schulz et al. 2013). All of which have direct or indirect antibacterial properties. But the question remains; how effective are these defence mechanisms?
A very simple and effective principle in testing the efficacy of an antibacterial agent is analysing the maximum bactericidal dilution (MBD). In essence, bacteria are grown in various dilutions of the antibacterial agent. The largest dilution (or lowest agent concentration) at which bactericidal activity is displayed is the MBD. The reciprocal of MBD is known as the bactericidal titre (BT). It is possible that the antibacterial efficacy of tears and saliva will vary between individuals.

Aim:
To determine the maximum bacteriolytic dilutions and bactericidal titre of tears and saliva from a number of students in order to: evaluate the antibacterial effectiveness and protective ability of these fluids and examine any variation in their efficacy between students.

Results:
Graph 1 (Tear BT): 6 students obtained a BT of 1000, 4 students obtained a BT of 10 and 70 students obtained a BT of 100. Unfortunately tears did not show any antibacterial activity against Branhamella. However, this could be due to the differences in cell wall morphology between gram negative and gram positive bacteria.
Graph 2 (Saliva BT): 1 student obtained a BT of 10000, 5 students obtained a BT of 10 and 3 students obtained a BT of 100. The saliva of 2 students also showed a BT of 10000 against Branhamella.

Discussion:
The aim of this experiment was to determine the maximum bacteriolytic dilution and bactericidal titre of tears and saliva taken from a large number of students in order to evaluate the antibacterial effectiveness of both tears and saliva and observe any variation in their efficacy between students. An analysis of MDP’s was used to generate histograms of BT’s in order to satisfy this aim.
The tears of some students (BT = 1000) were up to 100 times more effective than other students (BT = 10). The saliva of one student (BT = 10000) was up to 1000 times more effective than other students (BT = 10). Since the magnitude BT’s varied immensely, it is possible that certain students have a greater capacity to defend against bacterial infections than others. The frequency of tear BT’s however showed little distribution, as 87.5% of students obtained a BT of 10 (Table 1). When the distribution of BT frequencies in Graph 1 and Graph 2 are compared, it is unclear whether tears or saliva has stronger antibacterial activity.
Student’s tears showed consistent antibacterial activity against the gram-positive Micrococcus but not the gram-negative Branhamella. A possible explanation could be seen in the bacterial call walls. Unlike gram-positive bacteria, the peptidoglycan layer in gram negative bacteria lies between an outer an inner membrane. The outer membrane made up of lipopolysaccharides and phospholipids that protect the inner peptidoglycan layer from interacting with enzymes in the extracellular environment such as lysozyme, lactoferrin, secretory IgA and lipocalin (Junqueira, 1998).
Saliva bactericidal activity was demonstrated against gram-negative Branhamella. However, since only 2 out of ten students using saliva obtained this result, it is more likely that some form of experimental error has occurred which has corrupted the result (perhaps contamination of the saliva sample during lunch). It is clear that both tears and saliva have antibacterial activity against