J. C:OMP.
P.ATH.
1976.
\-OK.
86.
EXPERIMENTAL
STAPHYLOCOCCAL IN THE MOUSE
M.4STITIS
A MORPHOMETRIC STUDY OF EARLY CHANCES MAMMARY GLAND STRIICTITRE
1. Izl. REID,
R. D. HARRISON
and.J.
.4~ricultural ResearchCouncil, Institute jbr Researchon Animal Direuser.
IN
described (Anderson and Chandler, 1975) and inoculated (1010 organisms) into the 4th mammary gland on each side (R4 and L4) as described by Chandler (1970 . Sequential examination. Twenty mice were inoculated with a 0.1 ml. suspension 01 strain BB at 0 h. and 10 mice were killed at 6 h. (6 h. treated group) and the remaining 10 were killed at 12 h. after inoculation (12 h. treated groupj. Fourteen mice wcw used as uninoculated, involution controls; 6 were killed at 0 h. (0 h. control). 4 at 6 h. (6 11. control) and 4 at 12 h. (12 h. control). Bacteriological procedures. At necropsy each L4 mammary gland was removed and ground in a Griffith tube with 5 ml. of sterile isotonic salme and a total viable crll count per gland was obtained (Miles, Misra and Irwin, 19383. Histological and ultrastructural procedures. The R4 mammary gland was removcsd from each mouse at necropsy, weighed and divided in two. One half was fixed in 12 per cent. neutral buffered formalin and embedded in paraffin wax. Sections 5 pm. thick were cut and stained by Gram’s method, by HE. and by Gordon and Sweet.‘> reticulin method. The remaining half was fixed in 1 per cent. osmium tetroxide in phosphate buffer and embedded in Araldite for electron microscopy. Ten block3
330
I. M. REID et al.
were embedded from each animal. Sections, 2 pm. thick, were’ stained \vith toluidinc~ blue and examined in the optical microscope. Ultrathin sections \vt‘r(‘ C’LIL OII ;I Reichert OMU2 ultramicrotome. stained with uranyl acetate and Icad citralc, ;irrtl examined in an A.E.I. EM6 electron microscope. .lfoorphometric methods. These were applied to both histological and electron nli(.rt)scopic sections. The morphometric methods used were based on 11~~ point-countin,q method of Weibel, Kistler and Scherle ( 1966 j. Histological sections stained by the reticulin method of Gordon and Sbvrc.1 LV(YY used to estimate the number of acini per unit area. For each mouse. the number 01‘ 25 objective on the light microscope.. acini were counted in 10 fields using the The unit area was O-1089 mm.2. Thin (2 pm.) sections stained with toluidinc. blur For each animal. sections from 5 block\ were examined using the %:100 objective. were examined, 4 fields from each block. Using a multi-purpose test system of 80 points, the number of points lying on the following structures was counted !I interacinar tissue (2) acinar epithehum (3) lumen (4) fat in lumen (L fat in cspithelium and (6) neutrophils in lumen. In addition, the acinar \urf& arca pvr unit volume was calculated from the number of intersections of tht, acinus with tryi lines of total length 52 cm. At the ultrastructural level, thin sections from 5 blocks of each animal wcr( photographed in the electron microscope. Secretory cells were selected at random and photographed at a primary magnification of ~.‘6000. Five sections. 6 fields per section were photographed (for each mouse) giving a total of 30 photographs. The print size was 20 i 25 cm. and the final magnification / 18 000. Using a test system of 182 points the number of points falling on the following structures in the secretor\’ cell was counted (1) mitochondria (2) lysosomes (3) fat (4) Golgi vacuoles (5j Go@ membranes (6) rough endoplasmic reticulum (RER) (7) nucleus. ‘rhesr pomt counts were used to estimate the volume fraction of the lrarious components in the secretory cell. In addition, the surface area of RER was estimated by counting the number of intersections of RER with a test line of known length. The number of‘ mitochondria per picture was counted to calculate the number per unit area and hence the average \Tolume of an individual mitochondrion. Statistical analysis was performed by analysis of variance.
RESULTS
Bacteriology Six hours after inoculation, the number of viable staphylococci recovered from the L4 mammary gland expressed as a geometric mean (log,,) was 7.46 & 0.31 (s.D.). At 12 h. the number was 8.18 t_ O-71. Morphometrics of Mammary Acinus Natural involution. In control mice, involution for 12 11.resulted in a significant (P(O.05) increase in mammary gland weight but no change in the percentage of tissue occupied by acini (Table 1). There was, however, a significant (P
P.ATH.
1976.
\-OK.
86.
EXPERIMENTAL
STAPHYLOCOCCAL IN THE MOUSE
M.4STITIS
A MORPHOMETRIC STUDY OF EARLY CHANCES MAMMARY GLAND STRIICTITRE
1. Izl. REID,
R. D. HARRISON
and.J.
.4~ricultural ResearchCouncil, Institute jbr Researchon Animal Direuser.
IN
described (Anderson and Chandler, 1975) and inoculated (1010 organisms) into the 4th mammary gland on each side (R4 and L4) as described by Chandler (1970 . Sequential examination. Twenty mice were inoculated with a 0.1 ml. suspension 01 strain BB at 0 h. and 10 mice were killed at 6 h. (6 h. treated group) and the remaining 10 were killed at 12 h. after inoculation (12 h. treated groupj. Fourteen mice wcw used as uninoculated, involution controls; 6 were killed at 0 h. (0 h. control). 4 at 6 h. (6 11. control) and 4 at 12 h. (12 h. control). Bacteriological procedures. At necropsy each L4 mammary gland was removed and ground in a Griffith tube with 5 ml. of sterile isotonic salme and a total viable crll count per gland was obtained (Miles, Misra and Irwin, 19383. Histological and ultrastructural procedures. The R4 mammary gland was removcsd from each mouse at necropsy, weighed and divided in two. One half was fixed in 12 per cent. neutral buffered formalin and embedded in paraffin wax. Sections 5 pm. thick were cut and stained by Gram’s method, by HE. and by Gordon and Sweet.‘> reticulin method. The remaining half was fixed in 1 per cent. osmium tetroxide in phosphate buffer and embedded in Araldite for electron microscopy. Ten block3
330
I. M. REID et al.
were embedded from each animal. Sections, 2 pm. thick, were’ stained \vith toluidinc~ blue and examined in the optical microscope. Ultrathin sections \vt‘r(‘ C’LIL OII ;I Reichert OMU2 ultramicrotome. stained with uranyl acetate and Icad citralc, ;irrtl examined in an A.E.I. EM6 electron microscope. .lfoorphometric methods. These were applied to both histological and electron nli(.rt)scopic sections. The morphometric methods used were based on 11~~ point-countin,q method of Weibel, Kistler and Scherle ( 1966 j. Histological sections stained by the reticulin method of Gordon and Sbvrc.1 LV(YY used to estimate the number of acini per unit area. For each mouse. the number 01‘ 25 objective on the light microscope.. acini were counted in 10 fields using the The unit area was O-1089 mm.2. Thin (2 pm.) sections stained with toluidinc. blur For each animal. sections from 5 block\ were examined using the %:100 objective. were examined, 4 fields from each block. Using a multi-purpose test system of 80 points, the number of points lying on the following structures was counted !I interacinar tissue (2) acinar epithehum (3) lumen (4) fat in lumen (L fat in cspithelium and (6) neutrophils in lumen. In addition, the acinar \urf& arca pvr unit volume was calculated from the number of intersections of tht, acinus with tryi lines of total length 52 cm. At the ultrastructural level, thin sections from 5 blocks of each animal wcr( photographed in the electron microscope. Secretory cells were selected at random and photographed at a primary magnification of ~.‘6000. Five sections. 6 fields per section were photographed (for each mouse) giving a total of 30 photographs. The print size was 20 i 25 cm. and the final magnification / 18 000. Using a test system of 182 points the number of points falling on the following structures in the secretor\’ cell was counted (1) mitochondria (2) lysosomes (3) fat (4) Golgi vacuoles (5j Go@ membranes (6) rough endoplasmic reticulum (RER) (7) nucleus. ‘rhesr pomt counts were used to estimate the volume fraction of the lrarious components in the secretory cell. In addition, the surface area of RER was estimated by counting the number of intersections of RER with a test line of known length. The number of‘ mitochondria per picture was counted to calculate the number per unit area and hence the average \Tolume of an individual mitochondrion. Statistical analysis was performed by analysis of variance.
RESULTS
Bacteriology Six hours after inoculation, the number of viable staphylococci recovered from the L4 mammary gland expressed as a geometric mean (log,,) was 7.46 & 0.31 (s.D.). At 12 h. the number was 8.18 t_ O-71. Morphometrics of Mammary Acinus Natural involution. In control mice, involution for 12 11.resulted in a significant (P(O.05) increase in mammary gland weight but no change in the percentage of tissue occupied by acini (Table 1). There was, however, a significant (P
