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INTRODUCTION TO BACTERIOLOGICAL METHODS
04 04-08 Dec 2006; Laura Cinti - 12 Dec 2006

Antibiotics have been applied for centuries. Honey was for instance used by the Egyptians to dress wounds – and we know today that honey has an anti-bacterial effect drawing water from the bacterial cells. It was only in the 19th century that scientists became aware of how and why antibiotics were effective. The ability to harvest bacteria and use these to compete with pathogenic bacteria was one method trailed, another would apply chemical, and yet another enzymes. Perhaps the medical breakthrough came when Alexander Fleming in 1928 observed mould/fungi that had formed on a Petri dish inoculated with Staphylococcus causing inhibition to the bacterial growth. Curious, he identified the fungi as Penicillium notatum and cultured it further and finally injected one of his patients with it. The results where encouraging, but although his initial presentations were meet with scepticism; penicillium was brought into clinical use by the end of the 1940s.

Image from the History of the Public Health Service at the NIH website

Through the 1960s, new derivatives of penicillium were produced such as methicillin and ampicillin some killing both Gram-negative bacteria such Streptococcus spp. and Staphylococcus spp. and Gram-positive bacteria such as Escherichia coli. With the wide use of antibiotics in medicine today bacteria have now started to grow resistant to some of these new antibiotics – still new and smarter antibiotics are being produced. A last word on the relevance of antibiotics to recombinant DNA techniques lies in the ability to extract clean samples of the new organisms, i.e. as a result of having new genetic material added to an organisms DNA such as E.coli the new transgenic bacteria develops resistance to specific antibiotics – we can then add antibiotics to kill the non-modified leaving us with a pure sample.

Gathered at the Royal Free & University College Medical School, a group of eight researchers sit huddled over their lab bench awaiting the instructor. There is deathly silence. We are here to get an introduction to bacteriological methods which states in the program aims to provide researchers [working with projects involving bacteria/recombinant DNA work] with appropriate skills and knowledge to undertake basic bacteriology safely by good microbial practice.
 
Moving straight to the point Dr BM Charalambous [course organizer] provides us with an overview of bacteria. Derived from the Greek word meaning small stick – bacteria are unicellular microorganisms displaying wide range of morphologies and lack nucleus and membrane bound organelles.  Briefly, the different levels of laboratory containment were discussed. The levels of containment are parallel to the hazard group to which microbial agents have been assigned by the Advisory Committee on Dangerous Pathogens. As we were in a level 1 this required less rigorous controls than organisms belonging to level 4 (Ebola viruses and pathogens with no known cures.) 

the lab, © c-lab 2006
The lab


On this level it was rather we that constituted the danger for the organisms. The lab environment is a natural reminder that humans are a major source of contamination. 

During the course of the week several experiments were carried out.  These included methods for detecting antibiotic resistance (and identifying bacteria carrying antibiotic resistance), a mutagenesis experiment (and generate an antibiotic resistant mutant from a sensitive wild-type parent), gram-staining, and DNA transformation with a plasmid vector into Escherichia coli (and identifying the bacteria harbouring recombinant vectors). 

I guess I felt like the odd one out amongst the cocktail of researchers within science disciplines ranging from brain surgery, chemical bioengineering to chemistry - however this is not to say they always got it right (..and me always wrong). Apart from the antibiotics not always kicking in the right way – this is an area of waiting and hoping.

Experiment results, © c-lab 2006
Experiment results – my E.coli plate, the group plates stacked together, my mutagenesis plate with rifampicin resistant colonies.

 

Back in the lab we looked at two main methods for detecting antibiotic resistance: In the first, a culture is grown on a non-selective medium (not adding anything to inhibit or stop any particular organism growing) and then antibiotic is added via a plastic disc or strip. After incubation we could measure the zone of inhibition and compare with a standard. The second, used selective media (solid/liquid) incorporating a known concentration of antibiotics.

The Stoke’s Disc Sensitivity Method was the name of our first method and involved the preparation of iso-sensitest agar plates with control organisms inoculating the outer area of the plate. On the rotary plater I swabbed the centre of one plate (with shaky hands) with E.coli, another with Staphylococcus aureus (the same bacteria Flemings discovered mould on)  and the final plate with Streptococcus pneumonia - leaving a 2mm gap between these test and the control organism. The following antibiotic discs were applied; amoxicillin, chloramphenicol, colistin, cefotaxime, erythromycin, and penicillin.  The plates were left incubating for 18 hours at 37°C. 

My plates after incubation, © c-lab 2006
The Stoke’s Disc Sensitivity Method – my plates after incubation - on the left bad boys number one e.coli, in the center staphylococcus aureus and finally streptococcus pneumonia


After incubation we measured the zone radii of the test and control organisms in millimetres. This would tell us which of the antibiotic discs applied the test culture was resistant to and the degree of resistance. If the test organism grew up to the one of the discs we could be pretty sure it was resistant to this type antibiotics.  [Sensitive: zone radius not more than 3mm smaller than the control zone radius. Moderate:  zone radius at 3mm but not more than 3mm smaller than control zone radius.  Resistant: zone radius less than 3mm.]

The determination of Minimum Inhibitory Concentration (MIC) broth (second method), the bacteria (E.coli) were inoculated into test tubes with nutrient broth containing a series of diluted antibiotic concentrations.  The highest concentration was dispensed into the first test tube, then doubling dilutions were dispensed into the rest of the test tubes (2-11) in a receding fashion.  The final test tube (12) contained no antibiotics in the nutrient broth and hence used as a negative control. The MIC is the lowest concentration at which no growth is visible after overnight incubation at 37°C.  MIC confirms resistance of microorganisms to an antimicrobial agent.  We didn’t get it right the first time as the antibiotics were inactive – but the second time around I could see the culture growth stopped midways (at tube 8).

Minimum Inhibitory Concentration (MIC) experiment, ©: c-lab 2006
Determination of Minimum Inhibitory Concentration (MIC) experiment – my tubes with nutrient broth and dilution of antibiotic concentration.



The determination of Minimum Bactericidal Concentration (MBC) in broth was calculated by plating out the broth culture from each test tube (used in MIC) into an agar plate and incubated overnight at 37°C.  The MBC is the concentration at which no growth occurs.

 Minimum Bactericidal Concentration (MBC) experiment, © c-lab 2006
The determination of Minimum Bactericidal Concentration (MBC) experiment result [my plates 0, 1 & 2]

Minimum Bactericidal Concentration (MBC) experiment, © c-lab 2006
The determination of Minimum Bactericidal Concentration (MBC) experiment result [my plates 4, 8 & 16]


Minimum Bactericidal Concentration (MBC) experiment, © c-lab 2006
The determination of Minimum Bactericidal Concentration (MBC) experiment result [my plates 32 (eight colonies) & 64 (no growth on plate)]



Antibiotic susceptibility testing using E-tests, (first method) involved the use of commercially prepared strip to create a gradient of antibiotic concentration when placed on agar plates (inoculated with E.coli). This test is used as a quantitative technique for the determination of the MIC. 

E-tests experiment, © c-lab 2006
Antibiotic susceptibility testing using E-tests experiment – my plates before and after incubation.

Determination of cell counts by the Miles & Misra method involved the preparation of serial dilutions of bacterial suspensions.  The plates were divided into the number sectors and drops were deposited in the separate numbered sectors.  These were incubated for 18 hours at 37°C and observed for growth. 

Miles & Misra method, © c-lab 2006
Determination of cell counts by the Miles & Misra method – my plates after incubation.


For transformation of plasmid into E.coli experiment we used the TOPO TA Cloning Kit which was supplied with linearized vector [pCR2.1-TOPO], competent cells [One-ShotTM TOP10®], sequencing primers and reagents [except Taq] for control PCR cloning reaction and also [pUC18] plasmid DNA for transformation control.  Topoisomerase is an important class of enzyme which is involved in the supercoiling of the DNA making it topographical linearized and hence avoiding the enzyme to get knotted into the DNA during cleavage process (by having to circle around the DNA). The topoisomerase enzyme the binds to double-stranded DNA and cleaves it at a CCTTT cleavage site like a restriction endonuclease. The enzyme then remains covalently bound to the DNA and functions to relegate a DNA fragment with compatible overhangs, creating a recombinant molecule.

We mixed together 1µl microlitre (0.000001 litres) of fresh PCR product, salt solution and TOPO vector and 2 µl of sterile water (I dropped the lid….so wasn’t so sterile I suppose).  We incubated the mix at room temperature for 20 minutes, added 2 µl of this cloning mix to the competent cells and incubated on ice for 20 minutes.  We heat shocked the cell at 42°C for 30 seconds and added 250 µl of SOC medium and shook at 37°C for an hour (our lunch break).  After lunch we spread both 10 µl and 50 µl on the Ampicillian plates.  I battled getting the 10 µl onto the plate on the rotary plater – the drop kept sliding up and I couldn’t see [mental note: eye appointment], it worked after the third attempt. We incubated these plates.  The following day - only few of the plates managed to form colonies – a total of 4 colonies in the group.  On mine, there was nothing.  

Transformation of plasmid into E.coli experiment, © c-lab 2006
Transformation of plasmid into E.coli experiment


Gram-staining procedure
is used for classification of bacterial species.  The thick layers of peptidoglycan in the Gram-positive cell wall stained purple and the thin Gram-negative cell wall appears pink. We watched one of the lab research assistant carry out this rather messy procedure.  One member of our group volunteered to carry out this procedure.  We then looked at these gram stained bacteria in the microscope.

Gram-staining procedure, © c-lab 2006
Gram-staining procedure


It was great to meet the people in my course and hear about their research over coffee.  As for the event itself, the experiments took quicker than expected and my right little finger, in particular, had a good workout.

 

Other References: Advisory Committee on Dangerous Pathogens
Gram-staining procedure
TOPO® Cloning Technology
TOPO TA Cloning®
Peptidoglycan
Streptococcus pneumonia
Escherichia coli
Streaking for Isolation of Bacterial Colonies on an Agar Medium
Staphylococcus aureus
MacConkey agar
Amoxicillin
Chloramphenicol
Colistin
Cefotaxime
Erythromycin
Penicillin
Topoisomerase
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