“THE SENSITIVITY OF STAPHYLOCOCCUS AUREUS BACTERIA ON KAFFIR

LIME (CITRUS HYSTRIX) FRUIT EXTRACT”

I. METHODOLOGY

Extraction
Press the peels or maybe squeeze alot at once then the oil must be released. You
could.

Sensitivity Test

Gather the materials for culturing of the bacteria and for the sensitivity test. For the
culture media, we need Blood Agar Plate, Mueller Hinton Agar, stocked culture of
Staphylococcus aureus bacteria,wire loop or wire needle, alcohol lamp,sterile swab,
sterile NSS (Normal Saline Solution) and surgical gloves. For the preparation for the
anti bacterial medium we need, kitchen knife, Kaffir lime, sterilized dropper, strainer,
and small sterilized container such as test tube.

In preparing the bacteria for culture of Staphylococcus Aureus bacteria, begin by

getting your inculum using a sterile wire loop or wire needle. Pick a colony and streak
on your blood agar plate using three (3) quadrant streaking , then label the plate.Place
the plate inside the incubator for 24 hours at 37 degrees celcius.On the following day,
check for the growth, do catalase test and coagulase test and when both results are
positive, you then have your cultured Staphylococcus Aureus Bacteria, after this,
proceed to sensitivity test.

In sensitivity test, first warm your MHA (Mueller Hinton Agar) plate then flame your
wire loop until red hot, then cool it. Using the wire loop, pick a colony of
Staphylococcus Aureus Bacteria from the Blood Agar Plate or your cultured
Staphylococcus Aureus Bacteria, and make a suspension using your 0.9% sterile NSS
(normal saline solution) .Compare the turbidity with 0.5 McFarland standard. Dip the
sterile swab into your bacterial suspension and then streak on MHA (Mueller Hinton
Agar) plate three times, still in quadrant streaking but this time nearer streaks, leaving
no space. Stand for 10 minutes, then put a single drop of lime concentrate at the
center of the plate.Place inside the incubator at 37 degrees celcius for 24 hours.The
following day check for zone of inhibition by,measuring it with a vernier caliper from
the edge of the disk to where the growth begins. It is to be measured in mm units.
Boll Ist Sieroter Milan, 1975 Oct 20, 54(4), 300 - 4
{Evaluation of the lysozyme and DNAase activity for the identification of
pathogenic staphylococci}; Cava L et al.; The sensitivity of two tests recently
proposed for the classification of pathogen staphylococci were evaluated:
--production of DNA-ase with the modified method of Lachica et al.; --production of
lisozyme . The two above tests were studied with other six tests on 1,000 strains of
staphylococci showing a very high specificity . The Authors propose that the DNA-
ase production and the lisozyme production, also for their very simple execution,
should become routine tests to identify the strains of pathogenic staphylococci.

Lancet, 1975 Oct 4, 2(7936), 650 - 3

Methicillin-resistant staphylococci 1965-75; Kayser FH; Methicillin resistance in
Staphylococcus aureus has been one of the major problems of gram positive
infections in hospitals in the Zurich area . Up to 1971, about 20% of staphylococcal
disease was caused by these peculiar organisms . Since 1972, however, a gradual
decrease in the number of methicillin-resistant organisms has been observed, with
an unprecedented low of 3% in 1975 . The nearly 700 methicillin-resistant cultures
that have isolated since 1965 exhibited, with rare exceptions, conventional group-II
patterns of lysis in phage-typing and similar antibiotypes . It is suggested that all
these isolates are derivatives of a strain which has long existed in the staphylococcal
population . The reasons for the changes in the frequency of this strain as an agent
causing staphylococcal disease are unclear . The use of penicillinase-resistant beta-
lactam antibiotics in hospitals does not seem to play a major role in the distribution
and spread or in the disappearance of this strain.

J Hyg (Lond), 1975 Oct, 75(2), 259 - 74

Comparison of two methods for assessing the removal of total organisms
and pathogens from the skin; Ayliffe GA et al.; A standard hand-wash sampling
technique was compared with a simple finger-streak sampling method in assessing
the relative effectiveness of a number of alternative preparations used for
disinfecting the surgeon's hands (alcoholic 0.5% chlorhexidine, alcoholic 0.1%
tetrabrom-o-methyl phenol, a 4% chlorhexidine detergent solution, aqueous 0.5%
chlorhexidine, 2% 'Irgasan' detergent solution and, as control, bar soap) . There was
a fairly good correlation between the results of assessment by the two methods after
a single disinfection and after six disinfections, three on one day and three on the
next . Significant differences were shown in 21 comparisons between treatments
when the hand-wash sampling test was used, and 16 of these comparisons also
showed a significant difference by the finger-streak test . Staphylococcus aureus
was found in hand samplings from 5 out of 8 nurses in the Burns Unit of Birmingham
Accident Hospital by the hand-wash sampling method and from 2 of the same 8
nurses by the finger-streak method; the numbers were small, and no Staph . aureus
were isolated from the same hands after 1 min . wash in 70% ethyl alcohol . Similar
sampling on 29 nurses in other wards showed Staph . aureus on 3 nurses (one in
large numbers) by the hand-wash technique and on 1 nurse by the finger-streak
test; in only 1 nurse whose hands showed Staph . aureus before disinfection was the
organism found, by hand-wash sampling, after disinfection . Parallel sampling of
nurses' hands after washing with soap and water and after disinfection with 95%
ethanol showed larger numbers of Staph . aureus in a hospital for skin diseases than
in a general hospital, and a lower incidence and somewhat lower density of Staph .
aureus after ethanol treatment than after washing with soap and water; Gram-
negative bacilli, on the other hand, were commoner on hands in the general than in
the skin hospital, and present in much smaller numbers after disinfection with
ethanol than after washing with soap and water . Antibiotic sensitivity tests showed
the frequent recurrence on the hands of some nurses of multi-resistant Staph .
aureus with resistance patterns similar to those found in infective lesions in some of
the patients; different sensitivity patterns were usually found in staphylococci
isolated from the nose . Even in wards where many patients were infected, carriage
by nurses' hands of a particular strain of Staph . aureus did not seem to last for
more than a few days.

Antibiotiki, 1975 Oct, 20(10), 907 - 11

{Antibacterial activity of a lysozyme-like enzyme from staphylococci};
Afanas'eva TI et al.; A lysozyme-like enzyme isolated from the culture broth of Staph
. aureus 712 presented in its native state a lipoproteid complex . The lytic and
antibacterial spectrum of the enzyme was similar to that of the egg albumin
enzyme . However, the lipoproteid complex was somewhat superior to the egg
albumin complex in its activity against Micrococcus lysodeikticus, a number of
gramnegative bacteria and staphylococci . Out of the organisms studied Microccus
lysodeikticus, Sarcina and Bac . subtilis proved to be most sensitive to the both
enzymes . Comparison of the amount of the enzyme formed in the culture medium
with its minimum inhibitory concentration with respect to most of the microbes
tested provided a supposition that the lipoproteid enzyme did not play a significant
role in the process of the microbial antagonism.

Zh Mikrobiol Epidemiol Immunobiol, 1975 Oct, (10), 59 - 60

{Sensitivity to polyvalent therapeutic staphylococcal bacteriophage as a
supplementary criterion of staphylococcal pathogenicity}; Fel'dman IuM et
al.; It is recommended to use the capacity of pathogenic staphylococci to be lysed
by polyvalent therapeutic staphylococcal bacteriophage in the capacity of an
additional simple and accessible criterion of staphylococcus pathogenicity . Of 147
strains of the pathogenic plasmacoagulating staphylococci 101 were lysed by the
phage and of 166 nonplasmocoagulating nonpathogenic strains--only 6 . This test
correlated with the other signs of staphylococcus (lecithinase and hemolytic
activity) . The simplicity and sufficient specificity of this test permits to use it in any
practical laboratory . Polyvalent diagnostic phage can be used on the basis of
therapeutic bacteriophage by its additional adaptation to the pathogenic strains of
staphylococcus.

Rev Ig Bacteriol Virusol Parazitol Epidemiol Pneumoftiziol Bacteriol Virusol Parazitol

Epidemiol, 1975 Oct-Dec, 20(4), 219 - 30
{Studies of oral microbiocenosis . I . Incidence and properties of staphylocci
in young persons}; Sefer M et al.; The study of staphylococci in the buccodental
washings of 117 second year students at the Faculty of Stomatology, all healthy
youths aged 20-25 years, gave the following results: (I) stapphylococci were present
in 90% of the students; (II) 50.66% were oral carriers of pathogenic staphylococcal
strains; (III) in 23 cases two or more staphylococcal strains were found with
different metabolic and biologic properties; (IV) part of the bucal lysozyme was of
microbial origin since 95% of the coagulasopositive strains and 88% of the
coagulasonegative strains produce lysozymes; (V) 35.23% of the strains yield
staphylococcal active with regard to M . lysodeicticus; (VI) 56.19% of the
staphylococcal strains were resistant to penicillin and/or tetracyclin, and 5
coagulopositive strains developed in media with a penicillin concentration of 800
u/ml; (VII) 85% of the coagulopositive staphylococci were sensitive to human anti-
staphylococcal bacteriophages, the most frequent being phage type 77 of group III.

Tijdschr Diergeneeskd, 1975 Sep 15, 100(18), 991 - 4

{Staphylococci in cheese made from raw milk . (author's transl)}; Mol H et
al.; The presence of markedly varying numbers of coagulase-positive staphylococci
per gram (less than 10(2) -- greater than 10(6)) of cheese made from raw milk is
reported . In twenty cases, the strains isolated from the cheese were examined for
their ability to produce toxins . Eight strains were found to produce toxin A, no toxin
being isolated from cultures of the twelve other strains . In two cases, the studies
were done because of food poisoning which had probably been caused by the
cheese . The number of staphylococci was more than 10(6) in one case (cheese
about four weeks old) and approximately 1.5 x 10(4) in another (matured cheese) .
Subsequent studies showed that rather large amount of enterotoxin A were present
in the second cheese . The first cheese was not examined for the presence of toxin.

Biull Eksp Biol Med, 1975 Sep, 80(9), 59 - 61

{Identification of proteases of staphylococci by means of an antitrypsin
precipitating serum}; Krivoshein IuS et al.; Specific antitrypsin precipitating sera
obtained by intraconjunctival immunization of rabbits with crystalline trypsin was
used to reveal in the supernant of staphylococcal cultures protease having antigenic
character common with that of trypsin.This is confirmed by the trypsin accumulation
in the process of staphylococcus cultivation, by the inhibition of the proteolytic
activity of the antitrypsin precipitating sera and by the production of protein-
precipitate as a result of incubation of the supernant with the antitrypsin
precipitating sera.

Antibiotiki, 1975 Sep, (9), 823 - 5

{Effect of trypsin on the activity of tetracycline, erythromycin and
levomycetin}; Belizhenko GG; Trypsin had an antimicrobial effect on Staph .
aureus . Increasing of protein in the broth culture of the staphylococci was
inhibited in the presence of 50 gamma/ml of trypsin by 42.8% . The activity of
tetracycline, erythromycin and levomycetin increased, when they were used in
combination with trypsin, the effect of the trypsin combinations with tetracycline or
levomycetin was additive, while that of the combination with erythromycin was
synergistic.
Arch Dermatol, 1975 Sep, 111(9), 1135 - 9
Toxic epidermal necrolysis . A review of 75 cases in children; Rasmussen JE;
From 1965 to 1973, 75 patients were admitted to the Children's Hospital of Buffalo,
with toxic epidermal necrolysis (TEN) . A review of their records show an equal ratio
of males to females, with 73 whites and only 2 blacks . No patient was septic with
staphylococci, and there was only one death . A comparison of treatments indicates
that the disease is probably self-limited and not influenced by antibiotics or
corticosteroids.

Am J Clin Pathol, 1975 Sep, 64(3), 372 - 7

Frequency and some properties of clinical isolates of methicillin-resistant
Staphylococcus aureus; Blackwell CC et al.; Of 420 Staphylococcus aureus
isolates, 3.1% were methicillin resistant . Most of the 13 isolates were from the flora
of hospitalized patients . The organisms were also resistant to nafcillin and
cephalothin . They shared many of the properties with methicillin-resistant
staphylococci accumulated from other sources except for the lack of lysozyme-like
activity.

Zentralbl Bakteriol {Orig A}, 1975 Aug, 232(4), 446 - 53

{Influence of "clumping factor" from Staphylococcus aureus on
phagocytosis (author's transl)}; Hasche KD et al.; In phagocytosis experiments
the pathogenic significance of the "clumping factor" (CF) of Staphylococcus aureus
was studied . CF-positive staphylococci were engulfed considerably less by
leukocytes from rabbits and cattle than CF-negative staphylococci . Equally, the
engulfment of staphylococcal skeletons (SSk) coupled with partially purified CF was
significantly less than that of the SSk . The SSk had been prepared by extraction of
staphylocci with formic acid . By this method the SSk were freed of all substances
associated with staphyloccal virulence . Before coupling with CF the SSk were
activated by cyanogen bromide . These in vitro-observations could be confirmed by
the corresponding phagocytosis studies in experimental body cavities of rabbits .
Thus, CF apparently interfered with the engulfment of the staphylococci by the
leukocytes.

Zh Mikrobiol Epidemiol Immunobiol, 1975 Aug, (8), 67 - 71

{Quantitative indices of lysozyme activity in staphylococci}; Akatov AK et al.;
The lysozyme activity of 354 lysozyme-positive and 100 lysozyme-negative (by the
results of qualitative test) staphylococcus strains were studied quantitatively . The
method was based on titration of the lysozyme in the culture fluid of 48-hour broth
cultures of the strains under study . The quantitative method proved to be more
sensitive than the qualitative one, and permitted to reveal the lysozyme production
in 71% of the strains which were formerly considered to be lysozyme-negative .
There were distinct species differences between the lysozyme-positive
staphylococci: the mean lysozyme level in the S . aureus was significantly greater
then in the S . epidermis . There was no regular association between the lysozyme
activity, staphylococcus origin, bacteriophage reference and the antibiotic
resistance.

Zentralbl Bakteriol {Orig B}, 1975 Aug, 160(6), 551 - 67

Investigations on the efficacy of surface disinfection and surface cleaning
procedures . 1 . Tests under real-life conditions; Dupre M; The suitability of
disinfection preparations is assessed on the basis of laboratory tests, different
methods being used in the various countries . Since such model tests are rather
inadequate when it comes to judging surface disinfectants, additional in-use tests are
desirable . They might, in any case, serve as a reference system for judging the
evaluation criteria which still differ widely at the moment . The experiments
described in this study were chiefly designed to establish the effect of cleaning and
disinfection measures on bacteria normally present on surfaces and on the artificial
contamination of surfaces with Sarcinae as model germs . The tests were carried out
in the halls on 5 floors of a medical (lift landings) . "Rodac" plates were used to
identify the germs . 3 disinfectants (aldehydes, phenol derivative), 3 disinfectant
cleaning agents and soft soap were used . The preparations reduced the normal
germ count by approx . 80 per cent . The reduction was mainly due to the cleaning
effect (soft soap was as effective as the preparations with disinfectant properties) .
The effect on the "normal germ count" cannot, therefore, be used as sole criterion of
disinfectant action . When the various preparations were applied in twice the
concentration recommended for Staphylococcus hospitalism, the Sarcina count was
reduced by 99 to 99.9 per cent within 2 hours . The efficacy of disinfectants and
disinfectant cleaning agents was practically the same . Additional laboratory tests are
necessary before the effect of soft soap can be finally assessed . In actual practice
the unit count of pathogenic germs- such as Staphylococci and Klebsiellae- is too
low to enable an objective assessment of a disinfectant to be made . On the other
hand, artificial contamination with such pathogens is not possible because of the risk
involved . The use of Sarcina lutea as test germ is therefore subjects to certain
limitations . One of the prerequisites for using it is, for example, prior reduction of
the normal germ count to values of less than 500/100 cm2 . The second
communication will report on investigations into the chemoresistance of Sarcina and
how this compares with that of Staphylococcus aureus and Klebsiella . The need for
such studies arose from our present investigation.