COSTO DE LOS CASTILLOS DENTRO DE LAS FESTIVIDADES

8 . 1 Materials used to Compare the Activity Spectrum of Bovine Seminal Plasma with those of Human Seminal P lasma and Selected Antimicrobial Agents

Bovine seminal plasma was collected from vasectomised bulls as reported in CHAPTER 4 . 1 , and human seminal plasma was obtained from a healthy

vasectomised volunteer.

The selected antimicrobial agents tested in conjunction with bovine and human seminal plasmas ( lysozyme , polylysine , salmine and spermidine ) ,are described in CHAPTER 7 . 1 .

8 . 2 Techniques for Determining the Bacteria.l Sensi ti vi ty Spectrum of Bovine and Human Seminal Plasmas and Selected Antimicrobial Agents

The bacteria used to determine the sensitivity patterns of micro­ organisms to bovine 'and human seminal plasmas and to selected antimicrobial agents are listed in TABLE II .

These cultures were maintained on brain heart infusion agar plates held at 4 ' C after 24 hours incubation at 37 ' C and subcultured every 14 days .

The sensitivity o f each bacterium was determined by the radial diffusion technique described in CHAPTER 4 0 4 . For those test organisms which grew poorly in citrated nutrient agar, 10% calf serum was added to the assay

medium. This did not affect the sensitivity of M. lysodeikticus to bovine

seminal plasma and was therefore cons idered a suitable addition to the growth medium for more fastidious bacteria . The Haemophilus sp . tested required supplements of 10 micrograms/cm3 hemin and 4 micrograms/cm3 NADH . This depressed the action of spermidine against M . lysodeikticus , but the

proved sensitive to this polyamine , so its response pattern was considered comparable with the other bacteria tested .

8 . 3 Techniques for Determining the Sensitivity of Mycoplasmas to Bovine and Human Seminal Plasmas and Selected An·timicrobial Agents

The mycoplasmas used to determine the inhibito�7 effects of seminal plasma and known antimicrobial agents are listed in TABLE II .

ORGANISMS USED TO DETERMINE MICROBIAL SENSITIVITY PATTERNS TO BOVINE SEMINAL PLASMA, HUMAN SEMINAL PLASMA, LYSOZYME , POLYLYSINE , SALMINE AND

SPERMIDINE Gram positive Bacteria

Arthrobacter globiformis Bacillus cereus

Bacillus cereus v . mycoides ( 2 ) B acillus licheniformis

Gram negative Bacteria Acetobacter sp . citrobacter freundii Enterobacter c loacae Escherichia coli ( 8 ) Escherichia dispar Haemophilus influenzae Klebsiella aerogenes Bacillus megaterium ( 2 ) Bacillus stearothermophilus Bacillus subtilis Corynebacterium diphtheriae

gravis Klebsiella edwardsii v. edwardsii

Klebsiella sp . Corynebacterium diphtheriae

intermedius

Corynebacterium diphtheriae mitis Corynebacterium hofmanni

Corynebacterium pyogenes Corynebacterium xerosis Lactobacillus . bulgaricus ( 2 ) Lactobacillus casei

Lactobacillus casei v. alactosus Lactobacillus casei v . rhamnosus Lactobacillus plantarum Leuconostoc citrovorum Leuconostoc mesenteroides Micrococcus luteus ( 2 ) Micrococcus lysodeikticus Mycobacterium phlei Nocardia asteroides Staphylococcus aureus ( 8 ) Staphylococcus epidermidis Streptococcus cremoris Streptococcus equi Streptococcus faecalis ( 3 ) Streptococcus lac tis

Streptococcus mitis ( 2 ) Streptococcus mutans ( 3 ) Neisseria flavescens Neisseria gonorrhoea Neisseria meningitidis Pasteurella haemolytica Proteus mirabilis Proteus morgani Proteus rettgeri Proteus vulgaris ( 2 ) Providence sp . Pseudomonas aeruginosa Pseudomonas flourescens Salmonella arizonae Salmonel la cholerae- suis Salmonella gallinarum Salmonella newington

Salmone lla typhimurium ( 2 ) Serratia marcescens

Shigella flexneri Shigella sonnei Vibrio anguil larum .t-1ycoplasmas

Mycoplasma arginini Mycoplasma arthritidi s Mycoplasma hominis ( 2 )

Gram positive bacteria cont ' d Streptococcus pneumoniae Streptococcus pyogenes Streptococcus salivaris Streptomyces sp . Mycoplasmas cont ' d Mycoplasma ovipneumoniae ( 2 ) Ureaplasmas ( 2 )

FOOTNOTE : Where more than one strain of the same species of bacteria was examined, the number of strains tested is given in parentheses .

Mycoplasma species were grown initially in FM4 broth (Frey et al 1968 ) , modified to contain 0 . 2% arginine hydrochlori de and using phenol red as a pH indicator . The agar well diffusion technique used to de�ect

growth inhibition by the test solutions , was carried out on plates of FM4 medium solidified with brain heart infusion broth containing 0 . 8% agar . For FM4 agar, a more concentrated broth was used, which on addition of

sterile BHI/agar solution gave a medium of the usual strength ( the usual dilution was 2 FM4 broth : I BHI/agar solution ) . An aliquot ( 0 . lcm3 ) of mycoplasmal broth culture was swabbed onto the surface of the test plates before the wells were cut into the agar . The procedure then followed that

described in CHAPTER 4 . 3 .

Ureaplasmas were grown and tested in u9 broth ( Shepard et al 197 0 ) .

In this medium growth is detected by a colour change from yellow to pink, indicating an increase in alkalinity due to the production of ammonia from urea . Thus metabolic inhibition or lack of growth is indicated by the

absence of a colour change . To investigate the effect of bovine seminal plasma on ureaplasmas the growth of these organisms in U9 medium diluted I : _I and 1 : 2 with seminal plasma was compared with controls diluted with

distilled wate r . Subculture t o fresh U9 broth after 7 days was used to determine the presence of viab le ureaplasmas . Culture onto BHI agar was used to detect the presence of any contaminating bacteria .

8 . 4 Techniques to Determine whether Bovine Seminal Plasma is Bacteriostatic or Bacteriocidal

Overnight cultures of bacteria in brain heart infusion (BHI ) broth were adjusted with fresh sterile BHI broth to give a reading of 20 in a Klett- Summerson colorimeter fitted with a blue filter (400 - 460nm) . This

11 . . d . 1 7 f ' . / 3

ce suspenslon contalne approxlmate ly IX 0 colony ormlng unlts cm •

Antimicrobial material isolated from bovine seminal plasma by adsorption onto DNA was added to the bacterial suspension to give a final concentration of

3 3

Smg/cm , and the mixture held at 2S ' C . Aliquots of O . lcm were removed,

diluted and plated out onto BHI agar at various time intervals to determine the number of viable colony forming units still present in the sample . All

plates were incubated at 37 ' C for 24 hours . A parallel control containing

no additions was used to record normal bacterial behaviour over the time interval stUdied.

This assay was later modified : the BHI media described above being replaced by nutrient broth and nutrient agar .

CHAPTER 9 : TECHNIQUES USED I N THE I SOLATION OF AN ANTIMICROBIAL